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Polypeptide composition and biosynthesis of the yolk proteins in the firebrat Thermobia domestica (insecta thysanura).

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Archives of insect Biochemistry and Physiology 9:299-312 (1988)
Polypeptide Composition and Biosynthesis of
the Yolk Proteins in the Firebrat,
Thermobia domestica (Insecta, Thysanura)
Andre Rousset, Colette Bitsch, and JacquesBitsch
Laboratoire d 'Entomologie, CNRS URA 108, Universitt! Paul-Sabatier, 31062 Toulouse, France
ion-exchange chromatography of crude ovarian extracts of the primitive
insect Thermobia domestica allowed the separation, in native conditions, of
major and minor vitellins of molecular weights of 300,000 and 430,000,
respectively. Their polypeptide subunits were determined by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunotransfer
using an antiserum prepared against major vitellin. This protein was resolved
into large (M, 166,000-212,000) and small (around M, 50,000) polypeptides.
Minor vitellin, on the other hand, exclusively contained small polypeptides
that are immunologically different from those of the major vitellin.
Vitellogenin polypeptides from the hemolymph of mature females exhibited
electrophoretic mobilities and immunological properties similar to vitellin
polypeptides. Pulse-chase experiments showed that the female fat body
synthesizes radioactive and immunoprecipitable proteins, whose polypeptide
pattern is close to that of the major vitellogenin. However, part of the primary
vitellogenic polypeptides, at M, 210,000 and 212,000, is rapidly processed to
M, 176,000 and 182,000 subunits. These two polypeptides, as well as the
precursors, enter into the composition of the major hemolymph vitellogenin.
Finally, processing of the still uncleaved 210,000-212,000 polypeptides takes
place in the ovary, which performs the same step of vitellogenin maturation
as the fat body.
Key words: polypeptides, immunology, pulse-chase, processing
INTRODUCTION
Many investigations have been performed on insects to characterize the
hemolymph vitellogenins, which are mainly synthesized by the fat body,
and the so-called vitellins, which accumulate in the maturing oocytes (recent
reviews in [l-41). A comparison between various insect species of several
Received May 23,1988; accepted September 28,1988.
Address reprint requests to Andre Rousset, Laboratoire d'Entomologie, Universite PaulSabatier, 118 route de Narbonne, 31062 Toulouse Cedex, France.
0 1988 Alan R. Liss, Inc.
300
Rousset et al.
orders has led to interesting considerations on the evolution of these proteins
[5]. Little information is as yet available for other groups, such as the primitive Thysanura (Apterygota). In adult Thermobiu dornesticu, previous investigations using native polyacrylamide gel electrophoresis demonstrated the
presence of three vitellins in crude ovarian extracts [6]. Three female-specific
proteins of the same electrophoretic mobilities were also detected in the
hemolymph and their fluctuations measured during the alternative previtellogenic and vitellogenic phases of the reproductive cycles that are synchronized with the permanent molting cycles. Further investigations were carried
It was
out using an antiserum prepared against the major vitellin fraction
demonstrated that the vitellogenin assigned to M,430,000 is immunologically
different from the group M, 240,000 + 300,000 (the major fraction). The
purpose of the present study was to obtain a better characterization of the
yolk proteins of T. domestics by determining their polypeptide composition
and to investigate their biosynthesis using pulse-chase experiments with fat
bodies incubated in vitro.
[n.
MATERIALS AND METHODS
Animals
Themzobiu dornesticu (Packard) were reared under constant temperature and
humidity (37°C and 80% rh). Investigations were carried out on 8-month-old
females, kept with males, and inseminated during the first 3 days of the 10day intermolt period [8]. Most results were recorded on day 3 of the intermolt
during the vitellogenic phase of the ovarian cycle [9]. Some experiments
were also performed on females during the previtellogenic phase (on days 7
and 10 of the intermolt period) and on males collected on days 3 or 10.
Ion-exchange Chromatography
Ovaries pooled from 200 inseminated females collected on day 3 were
homogenized in phosphate-buffered saline (0.05 M sodium phosphate, pH
7.6, 0.01% Triton X-100, 2 pglml aprotinin, 0.3 M NaC1). After centrifugation,
the supernatant was applied to a DEAE* Sepharose CL-6B column (Pharma-
cia, Sweden), as previously described
dry at -80°C.
[q.Lyophilized fractions were stored
Protein Determination
Protein determination was performed according to the method of Bradford
[lo] using BSA (Sigma Chemical Co.) as standard protein.
Collection of Samples and PAGE
Ovaries were obtained from C02-anesthetized females, rinsed in Ephrussi’s saline solution [ll] containing 1 mh4 PMSF, pooled and stored frozen
*Abbreviations: BSA = bovine serum albumin; DEAE = diethylaminoethylcellulose;PAGE =
polyacrylamide gel electrophoresis; PMSF = phenylmethylsulphonyl fluoride; SDS = sodium
dodecyl sulfate; TCA = trichloracetic acid.
Yolk Polypeptides in Thermobia domestica
301
at -80°C. Hemolymph samples (2 p1) were collected in glass micropipettes
after puncturing the neck, immediately diluted with the Ephrussi's solution
(1:2), centrifuged (15,OOOg, 10 min, 4"C), and then stored frozen at -80°C.
Pore-limited electrophoresis was conducted in nondenaturing polyacrylamide gradient slab gels (6-20%) as previously described [6]; about 5 pg of
proteins were run in each well. The electrophoresis system of Laemmli [l2]
was used for separation of the peptides in SDS. About 8 pg of proteins were
used in each well. Electrophoresis was carried out at 24 mA (constant current)
for 5 h at 20°C using 16-cm-long, 1-mm-thick, 6.5% slab gels, with 3%
stacking gel (SDS-discontinuous buffer system in Bio-Rad Protean apparatus). Several denaturing reagents were tested: 1 and 0.5 M 2-mercaptoethanol, and 0.1 and 0.05 M dithiothreitol solutions. The solution of 0.1 M
dithiothreitol gave the best results and complete denaturation without
artifacts.
Molecular weights were obtained by comparing the electrophoretic mobilities of protein standards. Pharmacia molecular standards used were thyroglobulin ( M , 669,000), ferritin ( M , 440,000), catalase ( M , 232,000), lactate
dehydrogenase ( M , 140,000), and BSA ( M , 66,000). SDS-PAGE Bio-Rad protein standards used were myosin ( M , 200,000), galactosidase ( M , 116,000),
phosphorylase B ( M , 92,000), BSA ( M , 66,000), and ovalbumin ( M , 45,000).
The absorbance of the Coomasie Brillant Blue stain of the protein bands was
recorded with a microdensitometer (type PHI 3, Vernon, France, with a
Wratten N.22 filter).
Immunological Procedures
Antiserum against the major vitellin fraction was prepared as previously
described [q.For qualitative investigations, the Ouchterlony double-diffusion test was performed using 1%agarose gel in 0.02 M barbital buffer, pH
8.6, for 2 days at 4°C. Analysis was carried out using 4 pg of crude ovary
extract and 1.5 pg of each fraction separated by DEAE chromatography. The
gels were dried, stained for 5 min in 0.1% amido black 10B (Merck), and then
destained in 7% acetic acid. For immunoblotting, 0.5 pg protein samples
were first separated by 6.5% SDS-PAGE, equilibrated in methanollTris-glycine buffer (1:4), and then electrophoretically transferred onto a nitrocellulose
membrane (0.125 A, 15 h, 4°C). The blot was quenched for 3 h at ambient
temperature in a blocking solution (0.05 M sodium phosphate, pH 7.6, 0.15
M NaCI, 5% dried milk), incubated with the antiserum, and revealed.
Immunoprecipitation of vitellogenic proteins in 0.2 ml samples was carried
out by adding antiserum (25 pl), major vitellin (10 pg) as carrier, and 0.8 ml
buffer (0.05 M sodium phosphate buffer, pH 7.8, 0.15 M sodium chloride, 1%
Triton X-100, 1% Na-deoxycholate, 1mM PMSF, 2 pglml Aprotinin), according to the method of Bownes and Nothiger [13].
Synthesis of Yolk Proteins by the Fat Body and Pulse-Chase Experiments
Fat body attached to the dorsal part of the abdominal wall was carefully
dissected out in cold saline. After rinsing three times, fat bodies of two
vitellogenic females were incubated in 300 p1 of M 20 medium (Gibco, France)
302
Rousset et al.
that was supplemented with [3H]amino acid mixture (Amersham, Buckingamshire, England). The concentration of the labeled amino acids in the
medium was adjusted to 0.1 pCilp1. In further experiments, fat bodies were
labeled for a 10-min pulse, then removed, immediately homogenized, or
transferred into vials containing 300 p1 of unlabeled medium for up to a 90min chase. Fat bodies were homogenized in 3-4 vol of the immunoprecipitation buffer in an all-glass Potter-Elvehjem homogenizer and then sonicated,
and the homogenate centrifuged at lO0,OOOg for 60 min at 4°C. Vitellogenic
proteins were immunoprecipitated either in the supernatant of the fat body
samples or in the incubation medium. Total proteins of the medium were
precipitated with TCA (10%) in the presence of 0.3 mglml BSA as carrier.
Measurement of Radioactivity
The radioactivity of a precipitated aliquot was counted using a liquid
scintillation system (Beckman LS 3801). The immunoprecipitate was also
dissolved in an SDS-mercaptoethanol buffer [14] and the polypeptides separated on SDS-PAGE and subjected to fluorography, according to Chamberlain [El. The dried gels were exposed to a Kodak film at -80°C for an
appropriate period (between 7 and 30 days) and routinely developed. The
radioactivity of each polypeptide was calculated by correlating the total
radioactivity of the immunoprecipitate with the optical density of the radiolabeled bands measured with a microdensitometer.
RESULTS
Purification of Vitellins
Purification of vitellins was carried out using ion-exchange chromatography on ovarian homogenates (about 4,000 follicles). Elution with a NaCl
gradient separated several different fractions (Fig. 1) with absorbance values
from 0.2 to 0.8.
The fractions were analyzed on native PAGE and their patterns compared
with those from crude ovarian extract (Fig. 2). No protein was detected in
fractions eluted at 0.10 or 0.15 M NaCl (data not shown). The fraction eluted
at 0.22 M NaCl showed a single protein band, which exhibited the same
electrophoretic mobility as the band at M , 430,000 found in the ovarian
extract. Two protein bands were stained in each of the fractions eluted at
0.25 and 0.30 M NaCl; they corresponded to 240,000 and 300,000 vitellins.
The 300,000 vitellin band represented 80% of the proteins eluted at 0.30 M
NaCl, but only 40% of the proteins eluted at 0.25 M NaCI. Although separation between the 240,000 and 300,000 proteins was not complete, it appears
that the fraction eluted at 0.30 M NaCl contained most of the 300,000 vitellin
that, in the ovary, represents about 70% of the total soluble proteins.
Fractions were submitted to a double-immunodiffusion test using antiserum prepared against the protein fraction eluted at 0.30 M NaCl [q.As
shown in Figure 3, precipitation arcs were obtained against the crude ovarian
extract and also against the 0.30 and 0.25 M fractions. The continuity between
the precipitation arcs demonstrates the antigenic identity of the samples.
Yolk Polypeptides in Thermobia domestica
303
0
3
0.8-
0
E
0.6-
l 4
I
C
0
N
m
0)
0.4
-
u
C
a
a
;; 0.2ul
n
a
100
200
300
400
500
e l u t i o n volume
60 0
700
(mi)
Fig. 1. Elution profile of crude ovary extract of Thermobia domestica after DEAE chromatography. Fractions were eluted with a linear gradient from 0.05 to 0.40 M NaCl in 0.05 M
phosphate buffer, pH 7.6. Absorbance was measured at 280 nm, and 10-ml fractions were
collected.
Furthermore, the nonreactivity of the antiserum against the fractions eluted
at 0.10,0.15, or 0.22 M NaCl was observed in serial dilution tests, demonstrating the absence of the 240,000 and 300,000 vitellins in these fractions. Thus,
the chromatographic method used led to a suitable separation of two groups
of vitellins from the ovary of T. durnestica: a major vitellin that is chiefly
composed of the M, 300,000 protein, and a minor vitellin (M, 430,000).
Polypeptide Composition of the Vitellins
Protein fractions were analyzed by electrophoresis under denaturing conditions (Fig. 4) and the relative proportions of the polypeptide bands measured by densitometry (Table l).The major fraction, eluted at 0.30 M NaC1,
was resolved into numerous bands forming two groups: the first (about 75%)
composed of large polypeptides and the second (about 25%) composed of
small polypeptides. Six large polypeptides were detected: two thin bands at
M,210,000 and 212,000, forming less than 10% of the total polypeptides, then
three bands at M , 172,000, 176,000, and 182,000, among which the 176,000
and 182,000 bands were predominant and a 166,000 band was seen only as
traces. Two polypeptides of low molecular weight (46,000 and 57,000) were
also present. The fraction eluted at 0.25 M NaCl showed a polypeptide
pattern similar to that of the major fraction, with an increasing percentage of
the 166,000 band and also with smaller polypeptides of M, 50,000 and 52,000.
These three polypeptides are probably subunits of the 240,000 vitellin that is
predominant in the 0.25 M fraction. Lastly, the fraction eluted at 0.22 M NaCl
was resolved exclusively into small polypeptides with molecular weight
lower than 60,000, among which the 46,000 subunit was predominant. These
data can be compared with the results obtained from homogenates of vitellogenic ovaries (Table 1).More than 75% of the ovarian polypeptides were of
304
Rousset et al.
Yolk Polypeptides in Tbermobia domestica
305
high molecular weight, between 166,000 and 212,000. The bands at M , 176,000
and 182,000 together corresponded to about 65% of the total vitellogenin
polypeptides. Two additional bands of low molecular weight were present,
at M , 46,000 and 57,000. Thus, the polypeptide pattern from crude ovarian
extract is qualitatively and quantitatively similar to that of the fraction eluted
at 0.30 M NaC1.
Immunotransfer was carried out on these SDS-PAGE electrophoretograms
(Fig. 5). The minor 210,000 and 212,000 bands that were present in the ovary
extract and in .the 0.30 M fraction appeared to be highly immunoreactive. A
strong reaction was also shown for the bands between 166,000 and 182,000
in both the ovarian extract and the 0.30 M fraction. A reduction in crossreactivity was observed for the small polypeptides, particularly for the 46,000
and 57,000 bands. In contrast, the small polypeptides of the 0.22 M fraction
did not crossreact, indicating that the 430,000 vitellin and the 300,000 vitellin
polypeptides are different.
TABLE 1. Composition and Percentage of the Different Polypeptides From Female-Specific
Proteins of Vitellogenic Female Hemolyph (VQ H), From a Crude Ovary Extract (OV), and
From 0.30, 0.25, and 0.22 NaCl DEAE Chromatographic Fractions
Mr
x 10-3
212
210
182
176
172
166
57
52
50
48
46
44
VQH
ov
("/.I
("/.I
Large polypeptides
30
5
30
5
23
35
16
30
a
a
Small polypeptides
a
14
0.30
("/.)
4
5
20
32
12
a
0.25
0.22
("/.I
("/.I
1
12
8
16
15
13
11
15
12
a
10
13
20
11
56
21
'Traces.
Fig. 2. Native PAGE of crude ovary extract (OV) and of 0.30, 0.25, and 0.22 M NaCl DEAE
chromatographic fractions. ST = standard proteins (M, x
Fig. 3. Ouchterlony double-diffusion plate. Central well contained an antiserum against the
major vitellin fraction. Peripheral wells contained an OV and 0.30, 0.25, 0.22, 0.15, and 0.10 M
NaCl DEAE chromatographic fractions.
Fig. 4. SDS-PACE of the hernolymph of 7-day-old previtellogenic females ( P V Q H), 3-day-old
males (uH), and of vitellogenic females (V Q H), and also of O V and of 0.30, 0.25, and 0.22 M
NaCl DEAE chromatographic fractions. ST = standard proteins. The molecular weights ( x
of the hernolymphatic nonvitellogenic polypeptides are noted on the left; those of
vitellogenins and vitellins, on the right.
306
Rousset et al.
@
3:4
1:4
FBP
3:4
1:4
FB15
3:4
1:4
M15
Figures 5 and 6
3:4
1:4
FB30
3:4
1 :4
M30
Yolk Polypeptides in Thermobia domestica
307
Polypeptide Composition of the Hemolymph Proteins
The polypeptide composition of the hemolymph proteins was investigated
by SDS-PAGE analysis from samples collected from males and previtellogenic and vitellogenic females. The results are shown in Figure 4.
The polypeptide pattern of male hemolymph did not change throughout
the molting cycle. Two main groups of bands were discernible on the electrophoretogram. The first, constituting only 13% of the total polypeptides, was
composed of several closely spaced bands of high molecular weight (M,
between 169,000 and 230,000). The second group was composed of three
bands ( M , 75,000, 83,000, and 89,000), together representing about 80% of
the total polypeptides. Traces of other polypeptides, having a molecular
weight below 70,000, were also detected.
While the polypeptide composition of the previtellogenic female hemolymph was similar to that of males, in vitellogenic female hemolymph, the
band at M, 83,000 became very weak, the band at M , 182,000 was intensified,
and additional bands appeared at M, 176,000, 210,000, and 212,000, so that
the high molecular weight subunits were about 50% of the total hemolymph
polypeptides. The majority of the large polypeptides correspond to subunits
of proteins present in the hemolymph only during the vitellogenic phase of
the reproductive cycle. The distinct bands at M , 210,000 and 212,000 represented about 60% of the vitellogenic subunits, whereas the bands at M,
176,000 and 182,000 represented 40% (Table 1).
Immunotransfer did not detect reactivity against the polypeptides of the
male hemolymph (data not shown). By contrast, several polypeptides in the
hemolymph of vitellogenic females were highly immunoreactive, forming
two groups of bands at M , 210,000-212,000 and 176,000-182,000 (Fig. 5). The
immunoreactivity of the small polypeptides found in the hemolymph was
very weak.
Protein Synthesis by the Fat Body
Abdominal fat bodies were dissected out from firebrats collected on day 3
of the intermolt. Preliminary investigations involving incubation of fat bodies
for 3 h in the presence of labeled amino acids showed that male and female
fat bodies synthesized radioactive proteins that were continuously released
into the medium. Using SDS-PAGE and fluorography analysis, it was demonstrated that no radioactive polypeptide was detected in the immunoprecip-
(Figs. 5, 6) Fig. 5. lmrnunoblotting after separation by SDS-PAGE of 0.25, 0.22, and 0.30 M
NaCl DEAE chromatographic fractions of crude ovary extract (OV) and of vitellogenic female
hemolymph (V Q H). LP = large polypeptides. SP = small polypeptides.
Fig. 6. SDS-PAGE fluorographs of the imrnunoprecipitated proteins synthesized by the fat
body after an in vitro 3H-pulse-chase. FBP: fat body samples after 10-min pulse-labeling. FB15:
fat body samples after 15-min chase. M15: incubation medium samples after 15-rnin chase.
FB30: fat bodies after 30-min chase. M30: incubation medium after 30-rnin chase. Two lanes
are given for each sample: on the left, the lane obtained from 3/4 of the immunoprecipitate,
and on the right, that from the remaining 1/4.
308
Rousset et al.
itate of the incubation medium of male fat bodies, whereas several
radiolabeled subunits were detected in the immunoprecipitate of female fat
body medium: several large polypeptides (between M, 176,000 and 212,000)
were heavily labeled, while the labeling of other small polypeptides (about
M, 50,000) was very weak.
Two groups of labeled polypeptides were synthesized after a 10-min pulse
(Fig. 6): polypeptides ( M , 63,000, 225,000, and 280,000) whose radioactivity
was lower than 1%of the pulse-labeling and polypeptides more strongly
labeled whose molecular weight was similar to that of the major vitellogenic
polypeptides of the hemolymph (M, 176,000, 182,000, 210,000, and 212,000).
The total radioactivity of the immunoprecipitated proteins varied between
10,000 and 22,000 dpm depending on the samples, but the proportion between the different polypeptides was always similar: 75% for the doublet at
M, 210,000-212,000, 20% for the 182,000 band, and 5% for the 176,000 band.
The changes in the radioactivity of the different polypeptides were studied
after various times of the chase, and the results are expressed in Figure 7A
as a percentage of the total radioactivity of both fat body extracts and incubation medium. Polypeptides at M, 63,000, 225,000, and 280,000 disappeared
after 15-30 min (cf. Fig. 6). The radioactivity of the 210,000-212,000 bands
regularly decreased, whereas the 176,000 and 182,000 polypeptides progressively became heavily labeled. After 30 min, the 176,000 and 182,000 subunits
carried half of the total radioactivity, and at 60 min, each attained a radioactivity over that of the doublet at M, 210,000-212,000.
With regard to the immunoprecipitated proteins accumulating in the medium, it can be noted that the labeled 176,000, 182,000, and 210,000-212,000
bands were also present, whereas the 63,000, 225,000, and 280,000 polypeptides were absent (Fig. 6). A small radioactive polypeptide at M, 46,000 had
appeared in the medium after 20 min of incubation. During the first 10 min
of the chase, the radioactivity of the 176,000, 182,000, and 210,000-212,000
bands was distributed as in the fat body at time 0 (Fig. 78). Thereafter, the
radioactivity of the doublet 210,000-212,000 decreased markedly, while that
of the 176,000 and 182,000 bands increased. After 30 min of chase, the doublet
was less radioactive than either the 176,000 or 182,000 bands.
DISCUSSION
Previous work on the primitive insect Thermobiu domesticu has shown the
presence of several electrophoretically and immunologically distinct vitellins
in the ovary of adult females during the vitellogenic phase of their reproductive cycle [6,7l. In the present study, the vitellins extracted from mature
ovaries were separated into two main fractions: a major fraction in which the
300,000 vitellin was preponderant (80%) with a low percentage of the 240,000
vitellin, and a minor fraction in which the 430,000 vitellin alone was collected.
The polypeptide substructure of both fractions was then analyzed by SDSPAGE, and the crossreactivity of the subunits was detected using an antiserum prepared against the major vitellin fraction.
The major vitellin fraction contains both large and small subunits. The
high molecular weight polypeptides, 75% of the total polypeptides, are
Yolk Polypeptides in Thermobia domesfica
75 -
c
309
i
t\
210,000
212,000
\t;
\\
50 -
8
>
t
>
+
u
25
-
a
0
0
a
Lz
O -
!
0
I
,
,
/
5 10 15 20
60
30
90
C H A S E D U R A T I O N , rnin
75-
0-
3,
210,000
! 212,000
\
i\
\
50-
182~000
s
>
t
?
25-
V
'z
0
a
II:
0-
0
5 10 15 20
30
90
60
CHASE
DURAlION
, rnin
Fig. 7. Changes in the percentage of radioactivity of the main polypeptides from the vitellogenic proteins synthesized by in vitro cultured fat bodies at different times of the chase
following a pulse-labeling of 10 min. Other polypeptides, whose total radioactivity was below
I % , have not been represented. Time 0 denotes the beginning of the chase. A, Total vitellogenic proteins, both from fat body and from the incubation medium. B, Vitellogenic proteins
secreted into the incubation medium. Subunits at M, 176,000 (A ), 182,000 ( O ) ,and 210,000
and 212,000 (0).
composed of a "doublet" at M , 210,000-212,000 and of several bands from
166,000 to 182,000. Two small subunits of My46,000 and 57,000 constitute
25% of the total polypeptides. All the subunits are immunoreactive. The
minor vitellin fraction exclusively contains small polypeptides, among which
the most abundant ( M y46,000) did not crossreact with our antiserum. So the
small subunits of the minor vitellin (M, 430,000) appear to be immunologically different from those contained in the major vitellin, although their
electrophoretic mobility was found to be identical. The polypeptide pattern
310
Rousset et al.
from crude ovarian extracts is qualitatively and quantitatively similar to that
of the major vitellin fraction. The hemolymph of vitellogenic females contains
polypeptides of the same electrophoretic mobility and of the same immunological properties as those of the major vitellin fraction, but with different
proportions (Table 1).
On the basis of the size of their vitellogenin polypeptides, pterygote
insects have been distributed into three groups [1,4,5]. Although the major
vitellin fraction of T. domesticu (bands at M , 240,000 + 300,000) contains two
different classes of polypeptides, this insect belongs to group one, which
comprises the majority of insect orders, namely, the Dictyoptera and Orthoptera. But, by its minor vitellin (at M, 430,000), which exclusively contains
small polypeptides, it could also find a place in group three of Harnish and
White (or class I1 of Kunkel and Nordin), which, until now, seemed to be
restricted to higher Diptera. Thus, the primitive apterygote insect T. domestics, generally regarded as closely related to the hypothetical ancestor of
pterygote insects, appears to constitute an original model, characterized by
the coexistence of two distinct classes of vitellogenins.
Incubation of fat bodies of T. domestica in the presence of tritiated amino
acids demonstrated that the fat body synthesizes proteins that were precipitated by our antiserum. The polypeptide composition of the vitellogenic
proteins secreted by female fat bodies is characterized by high incorporation
of radioactivity in the large subunits. The pattern obtained after fluorography
appears to be very similar to that of the hemolymph polypeptides after
immunotransfer. Thus, the polypeptide composition of vitellogenic proteins
secreted by cultured fat bodies is close to that of the hemolymph vitellogenins.
Biosynthesis of yolk proteins by the fat body of 7: domesticu was also
investigated using pulse-chase experiments in order to learn about the processing of the major vitellogenin. The radioactivity of the doublet at M ,
210,000-2l2,000, which contains 75% of total radioactivity of fat body extracts
after a 10-min pulse, progressively decreases during the chase whereas the
radioactivity of the 176,000 and 182,000 subunits increases. In addition, a
46,000 polypeptide secreted into the medium becomes slightly radioactive.
These changes involve a transfer of radioactivity from the larger to the
smaller subunits and suggest that the 210,000-212,000 polypeptides are the
precursors, about a third of which is cleaved in the fat body. The uncleaved
precursors are then secreted into the hemolymph, along with their proteolytic products. Moreover, pulse experiments show that the female fat body
synthesizes some other polypeptides, at M, 63,000, 225,000, and 280,000,
which are not secreted into the medium. The very weak radioactivity of these
bands (below 1%) prevents a clear insight into their possible role in the
processing of the major vitellogenin. In the majority of insects, which fall
into group one of Harnish and White [1,5], vitellogenin appears to be synthesized as a large precursor with a molecular weight generally over 200,000
[16]. In Leucophuea maderue, for example, two large polypeptides of My179,000
and 260,000 have been suggested as the precursors of several smaller vitellogenins ranging from 57,000 to 118,000
However, different results have
been obtained in the same cockroach species by Brookes [18], who considered
that the large polypeptide (M, 250,000) is not a subunit of the vitellogenin,
[la.
Yolk Polypeptides in Thermobia domestica
31 1
although it precipitates with antiserum against the vitellin. In another recent
study on Leucophaea maderae, Della-Cioppa and Engelmann [191, using pulselabeling of fat body with 32P, found a phospholabeled 215,000 polypeptide
that precipitated with antiserum against vitellogenin. This pro-vitellogenin
was proteolytically processed within the fat body, and the products were
secreted into the hemolymph as mature vitellogenin subunits. In Locusfa
migvatoria, two primary products at M, 225,000 and 235,000 have been identified by Chen [20] and considered as vitellogenin precursors, while the final
polypeptides range from M, 52,000 to 126,000. According to Wyatt et al. [21],
the primary translation product of L. migratoria fat body is a 200,000 peptide,
whose glycosylation yields the two vitellogenic polypeptides of M, 225,000
and 235,000.
In T. domesticu, a comparison of the different vitellogenic polypeptides
from the hemolymph with those from mature ovaries reveals striking changes
in their respective percentages: the 210,000-212,000 bands were much less
abundant in the ovary (10 vs. 60%), the 176,000 and 182,000 bands much
more abundant (65 vs. 40%), whereas polypeptides smaller than 60,000
reached a quarter of total vitellins. These findings indicate an additional
processing phase of vitellogenins when they are sequestered by the ovaries.
Ovarian processing has been considered in some other insect species, such
as Leucuphaea maderae [19] or Blafella gemanica [22]. In Locusfa migrabria, it has
been suggested that the proteolytic cleavage of the vitellogenic proteins,
which normally occurs in the fat body, may be continued in the hemolymph
and in the ovary [21]. A recent study on Aedes aegypti [23], comparing the
peptide substructure of the vitellogenin and of the vitellin, also concludes
that the final processing of the yolk proteins takes place in the ovary. In T.
dornesticu, a similar step in the processing of the primary polypeptides occurs
both in fat body and in ovary tissues. This situation appears to be unique
among insects and therefore may have evolutionary significance.
LITERATURE CITED
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(1982).
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Thermobiu dornesticu. Physiol Entomol 10, 15 (1985).
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phosphorylated precursor. Insect Biochem 27, 401 (1987).
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vitellin and vitellogenin. Arch Insect Biochem Physiol4, 81 (1987).
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