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Development of male-incubated ovaries in the gypsy moth Lymantria dispar.

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Archives of Insect Biochemistry and Physiology 16:221-234 (1 991)
Development of Male-Incubated Ovaries in the
Gypsy Moth, Lymnntria dispar
JoanneBallarino, M i c h a e l Ma, Tsuey Ding, and Craig Lamison
Department of Entomology (1.B., M.M.)
and Center for Agricultural Biotechnology, Ma yland
Biotechnology lnstitute (M.M.), University of Ma yland, College Park, Maryland; Institute of
Zoology, Academia Sinica, Beijing, People's Republic of China (T.D.); and E.I. Du Pont De
Nemours & Co., Ag Diagnostics, Newark, Delaware (C.L.)
Ovaries from Lymantria disparfemales were transplanted into an environment
lacking vitellogenin, the male milieu, in order to determine how the presence of vitellogenin i n the hemolymph affects the process of protein uptake
by gypsy moth oocytes. When undeveloped ovaries from newly ecdysed last
instar females were transplanted into males of the same stage, follicles detached from the germarium and increased i n size, but the growth of oocytes
proceeded more slowly than those from female controls. Although chorion
formation was delayed i n male-grown ovaries, scanning electron microscopy
of chorionated eggs recovered from adult males showed that a chorion with
normal surface architecture was formed by the adult stage. SDS-PACE analysis of the male-grown ovaries and hemolymph from males receiving ovaries
showed that vitellogenin production was not stimulated by the organ transplant and only male hemolymph proteins were internalized by the maleincubated ovaries. Thus, in the absence of vitellogenin, endocytosis of male
hemolymph proteins occurred, but the rate of oocyte growth was slowed.
Key words: vitellogenesis, hernolymph proteins, transplanted ovaries, follicle growth
INTRODUCTION
In egg-laying species, the rapid growth of the oocyte is due predominantly
to the storage of large amounts of yolk over a relatively short period of time.
Acknowledgments: We thank Dr. Robert Bell of the Insect Reproduction Laboratory at USDA,
Beltsville, M D for providing us with gypsy moths. Timothy Mau el of the Laboratory for Biological
Ultrastructure at the University of Maryland provided help uI suggestions on the scanning
electron microscopy. Professor William Telfer (Univ. Pennsylvania), Professor M.L. Pan (Univ.
Tennessee), Michael Blackburn (Univ. Maryland), and Dr. David Dussourd (Univ. Maryland)
provided us with helpful comments on the manuscript. This research was supported by the
College of Agriculture and Life Sciences, University of Maryland at College Park, and by the
Maryland Experiment Station. This paper is Scientific Article No. A6061, Contribution No. 8223
and it i s contribution No. 57 from the Laboratory for Biological Ultrastructure, Universify of
Maryland.
B
Received July27,1990; accepted December 13,1990.
Address reprint requests to Dr. Michael Ma, Department of Entomology, University of Maryland,
College Park, M D 20742.
0 1991 Wiley-Liss, Inc,
222
Ballarino et al.
In most insects the yolk proteins, or vitellins, are synthesized not by the oocyte,
but by the fat body. The form synthesized by the fat body is termed vitellogenin,
and is secreted into the hemolymph and incorporated into the oocytes by
endocytosis. The timing and hormonal regulation of vitellogenic events is
correlated with the feeding habits of the adult stage insect. In insect species
which feed as adults, vitellogenesis generally takes place in the adult stage
[1,2], while in insects which do not feed as adults, vitellogenesis takes place
in the pharate adult stage [3-61.
The endocytotic uptake of vitellogenin appears to be receptor-mediated as
evidenced by the presence in the oocyte of clathrin-coatedpits and vesicles to
which vitellogenin adheres 17-91 and by kinetic binding studies demonstrating saturable uptake of radiolabeled vitellogenin by follicles cultured in vitro
[lo-151. Is the binding of the vitellogenin ligand to its receptor necessary to
the process of endocytosis in oocytes? In many membrane transport systems
which utilize receptor-mediated endocytosis to transport macromolecules, the
presence of the macromolecular ligand is not a necessary condition fox endocytosis to occur [16]. From the evidence provided by studies of ovarian development in the male milieu, this appears to be the case in several Lepidoptera
with nonfeeding adults 1171. Transplantation of ovaries into male Hyalophora
cecropia [18,19]and Bombyx mori [20,21]results in the uptake of male hemolymph
proteins in the absence of vitellogenins. Although the male-grown eggs are
less numerous in B. mori, they reach nearly normal size and can be stimulated
parthenogenetically to develop and hatch into larvae [22].
The gypsy moth, Lymantria dispar, does not feed as an adult, and like other
lepidopteran species with nonfeeding adults ( H . cecropia, B. mori),vitellogenin
synthesis and uptake occurs before adult emergence 1231. We have found, however, that the gypsy moth differs from other lepidopterans with nonfeeding
adults in the timing of follicle detachment, vitellogenin synthesis, and vitellogenin uptake [23]. Follicle detachment from the germarium begins on day 4
of the last larval instar (apolysis begins on day 8 of the last larval instar).
Vitellogenin production and secretion into the hemolymph is initiated even
earlier, on day 2 of the last larval instar in gypsy moths, notably prior to pharate
pupa formation. This is earlier than in B. mori in which the presence of
vitellogenin has been reported in the pharate adult stage [6] or H. cecropia in
which vitellogenin has been detected during the larvel-pupal molt [5,18].
Although vitellogenin synthesis begins early in the last larval instar in gypsy
moths, vitellogenin uptake does not begin until day 3 after pupal ecdysis [23],
when pharate adult development has begun. Since the final larval stadium
lasts approximately 10 days in female gypsy moths, and uptake does not begin until day 3 after pupal ecdysis, this results in a long period during which
vitellogenin titers are high in the hemolymph. Why does vitellogenin synthesis begin so early in the gypsy moth? Certainly there must be a cost to maintaining high vitellogenin titers over a long period of time. Is this prolonged
period of high vitellogenin titers in the hemolymph necessary for the onset of
patency and endocytosis? Does vitellogenin cue the onset of these processes?
Or would these processes proceed normally in the absence of vitellogenin?
We felt that answers to these questions might be provided by studying ovarian development in an environment lacking vitellogenin: the male milieu. Our
objectives for this study were to monitor possible differences in the develop-
Development of Male-IncubatedOvaries
223
ment of male-incubated ovaries with respect to 1)the number of folicles formed;
2) the size of the follicles (i.e., the degree of growth and uptake); 3) the formation of the chorion; and 4) the protein composition of mature eggs.
MATERIALS AND METHODS
Insect Rearing
Gypsy moths (L. dispur, NJSS-22 strain) were received as 4th stadium larvae
from Dr. R.A. Bell of the Insect Reproduction Laboratory at USDA, Beltsville,
Maryland. The larvae were maintained on a modified wheat germ diet [24] at
26 ? 1°Cunder a 16:8 L:D photoperiod.
Microsurgery
Ovarian cysts were removed from newly ecdysed last larval instar females
and transplanted into newly ecdysed last larval instar males, or into female
controls from which the ovarian cysts had just been removed. Larvae were
placed on ice prior to surgery and pinned down with 2 pieces of flexible tubing which were cut in half lengthwise. A window was cut from one of the
pieces of tubing and placed over the midpoint of the fifth tergum. A small
longitudinal incision was made at the midpoint of the fifth tergum and the
ovaries were removed and placed in Insect Ringer solution. A similar incision
was made at the fifth tergum in newly ecdysed last instar males and the ovarian cysts were implanted into the male abdominal cavity, or into the abdominal cavity of other females from which the ovarian cysts had been removed.
Wounds were blotted dry with sterile Kimwipesm, dusted with penicillin and
streptomycin, and sealed with dental wax.
Monitoring the Production and Growth of Ovarian Follicles
Male-grown, female-operated control and normal control ovaries were dissected from day 5 last instar larvae, from day 1, day 4, day 6, and day 8 pupae,
and from newly emerged adults. A minimum of 5 insects was used for each
time period and experimental group. Ovarian cysts or ovaries were removed,
and the ovarioles were dissected free of fat body, connective tissue, and ovariole
membranes, and observed under a dissecting microscope with oblique lighting. The number of follicles per ovariole that were detached from the germarium
was counted for each time period. A follicle was considered "detached" when
its opalescent oocyte lined up with the other oocytes like a string of pearls.
Growth of the ovarian follicles was monitored by measuring the diameters
of the terminal (closest to the oviduct) follicles (including oocytes, nurse cells,
and follicular epithelium) with an ocular micrometer, In addition, the number
of oocytes (not follicles) per ovariole which had reached an arbitrary width of
100 pm was counted for each time period. This number was converted to percent to eliminate the effect of the reduced number of follicles in transplanted
ovaries. This latter measurement was made to determine whether follicles other
than the terminal follicle exhibited a reduced rate of growth in male-grown
ovaries. For day 8 pupae and adults, the number of chorionated eggs was
counted, and the diameters of the terminal chorionated eggs were measured
with an ocular micrometer.
224
Ballarino et al.
Gel Electrophoresis
Adult ovaries from males, female-operated controls, and normal controls
were removed and the follicles were dissected free of fat body, connective tissue, and ovariole membranes. Follicles were washed 5 times in phosphatebuffered saline (137 mM NaCl, 8 mM Na2HP04, 1.5 mM KHzPO3, 2.7 mM
KCl, pH 7.4). They were then homogenized in phosphate-buffered saline with
1 mM PMSF" and saturated PTU, and centrifuged for 5 rnin at 12,OOOg. The
soluble supernatants were collected and frozen with PMSF and PTU at - 70°C
for no more than 1week. Previtellogenic ovaries from day 8 last larval stadium
of normal insects were prepared in the same manner.
Hemolymph was collected from day 1 pupae of normal males, normal females, males receiving ovary transplants, and female operated-controls receiving ovary implants. An incision was made in the pupal case at the first
abdominal segment and a Gilson micropipettor was used to collect the hemolymph. The hemolymph was added to microfuge tubes containing PMSF and
PTU, on ice, and was centrifuged at 4°C for 5 min at 12,OOOg to remove hemocytes. They were stored with PMSF and PTU at - 70°C for no more than 2 weeks.
SDS-PAGE was performed according to the methods of Laemmli [25]. For
each hemolymph sample, 1 pl of hemolymph was loaded. For each ovary extract sample, except the previtellogenic ovary, extract from 4 terminal (closest
to the oviduct) follicles of adult insects was loaded. For the previtellogenic ovary,
extract from 4 whole ovarioles from the 8 day last larval stadium was loaded.
Scanning Electron Microscopy
Chorionated eggs were dissected from males, female-operatedcontrols, and
normal controls and fixed in 4% glutaraldehyde in Millonig's buffer pH 7.3
[26] for 24 h at 4°C. They were washed 3 times in buffer and then post-fixed in
1% osmium tetroxide in Millonig's buffer for 1.5 h. The eggs were then dehydrated in an ethanol series, critical point dried, mounted on stubs, and coated
with gold-palladium. They were observed under an AMR 1820 scanning electron microscope at 20 kV.
RESULTS
After transplantation of ovaries, female operated-control and male larvae fed,
pupated, and eclosed, with a 20% mortality rate. At all stages, the number of follicles detached from the germarium per ovariole was lower in transplanted ovaries
from both males and female operated-controls when compared to ovaries from
normal females (Figs. 1, 2). There was no significant difference between males
and female operated-controls in the number of follicles detachediovariole.
The diameter of the terminal follicle (the follicle closest to the oviduct) increased at a greater rate in normal control ovaries and female operated-control
ovaries than in male-grown ovaries (Fig. 3). The terminal follicles from normal
ovaries were significantly larger than those from male ovaries at all stages except day 1 pupa. Terminal follicles from female operated-control ovaries were
significantly larger than those from male-grown ovaries at and following the
'Abbreviations used: k D = kilodalton; PMSF = phenylmethylsulfonyl fluoride; PTU = phenylthiourea; SDS = sodium dodecyl sulfate.
Development of Male-IncubatedOvaries
225
Fig. 1. Ovarioles dissected from normal control and male-grown ovaries at various times in
the pupal stadium. Male-incubated ovaries appear in A-D and normal control ovaries in E-H.
Ovariole age increases from top to bottom, with ovarioles from 4 day pupae shown in A and E,
from 6 day pupae in B and F, from 8 day pupae in C and G, and from newly emerged adults in
D and H. Bar length = 1crn. The bar i s shown in H, but is the same for all micrographs.
day 6 pupa. Prior to day 6, there was no significant difference between female
operated-controls and males in the size of the terminal follicle.
A greater percent of the oocytes in normal ovaries and female operatedcontrol ovaries reached an arbitrary width of 100 km than in male-grown ovaries (Fig. 4). This difference between normal and male-grown ovaries in the
percent of oocytes that reached a width of 100 pm was significant at and fol-
226
Ballarino et al.
-0- normal
200
-
150
-
100
-
0
5d L
-0-
- . A - -male
ld P
4d
P
op ctrl
64 P
8d
P
Adult
Developmental Stage
Fig. 2. The number of follicles detached from the germarium per ovariole as a function of
insect age. Each point represents the mean number of follicles detached per ovariole in ovarioles
from at least 5 different insects. At least 3 ovarioles were examined per insect. The bars represent the standard error around the mean. Where bars do not appear, the standard error was
smaller than the size of the marker. Significant difference from male-incubatedovaries, as determined with Student's t-test, i s indicated with an asterisk.
-0-
normal
- 0-
-.4-.
male
OD
ctrl
1.25
1.00
0.75
0.50
0.25
0.00
0
5d L
id P
46
P
6d P
8d P
Adult
Developmental Stage
Fig. 3. The diameter of the terminal follicle (the follicle closest to the oviduct) as a function
of insect age. Each point represents the mean diameter of the terminal follicle of ovarioles
from at lest 5 different insects. At least 3 ovarioles were examined per insect. The bars represent the standard error around the mean. Where bars do not appear, the standard error was
smaller than the size of the marker. Significant difference from male-incubatedovaries, as determined with Student's t-test, i s indicated with an asterisk.
Development of Male-Incubated Ovaries
-0- normal
56 L
- . A - . male
227
-0- op ctrl
4d P
66 P
Developmental Stage
Id P
8d P
Adult
Fig. 4. The percent of oocytes that reached a width greater than 100 pn as a function of insect age. Each point represents the mean percent of oocytes that reached a width greater than
100 p m in ovarioles from at least 6 different insects. At least 3 ovarioles were examined per
insect. The bars represent the standard error around the mean. Where bars d o not appear, the
standard error was smaller than the size of the marker. Significant difference from male-incubated
ovaries, as determined with Student’s t-test, is indicated with an asterisk.
-0-
normal
-.A-.
male
- 0-
op ctrl
r
0
Sd L
ld P
4d P
66 P
8d P
Adult
Developmental Stage
Fig. 5. The percent of chorionated eggs as a function of insect age. Each point represents the
mean percent of chorionated eggs in ovarioles from at least 7 different insects. At least 3 ovarioles
were examined per insect. The bars represent the standard error around the mean. Where
bars d o not appear, the standard error was smaller than the size of the marker. Significant
difference from male-incubated ovaries, as determined with Student’s t-test, is indicated with
an asterisk.
228
Ballarino et al.
lowing the day 4 pupal stage. The difference between female operated-control
and male ovaries was significant at and following the day 6 pupal stage. Percentages were used to eliminate the effect of the reduced number of follicles
in transplanted ovaries.
A significantly greater percent of chorionated eggs was found in normal
and female-operated control ovaries than in male ovaries at the day 8 pupal
stage and the adult stage (Fig. 5). In adult male-grown ovaries some ovarioles
contained chorionated eggs, while other ovarioles within the same ovary contained smaller follicles with no evidence of chorion formation at the light microscope level. Chorionated eggs from adult males reached 95.0% of the
diameter of normal chorionated eggs and 94.2% of the diameter of female
operated-control chorionated eggs. This difference between the chorionated
eggs of males and those of normals and female operated-controls, though small
(5% and 5.8% respectively), was significant. The variance in the diameter of
chorionated eggs was small within experimental groups, with a mean and
standard deviation of 1.20 + / - 0.03 mm for normals, 1.21 + / - 0.03 mm for
female operated-controls, and 1.14 / - 0.07 mm for males.
Scanning electron microscopy showed that the surface architecture of the
chorion of adult eggs is similar in males, female operated-controls, and normal controls (Figs. 6, 7). Both the micropyle region (Figs. 68, 78) and the
honeycomb-like region (Figs. 6C, 7C) developed normally in male-grown
ovaries.
SDS-PAGE analysis of hemolymph from normal females and normal males,
and from experimental females and males receiving ovary transplants’ showed
that vitellogenin production was not stimulated by the ovary transplant (Fig.
8; arrows indicate vitellogenin subunits). SDS-PAGE analysis comparing adult
male-grown ovaries to normal female larval ovaries (previtellogenic),normal
female adult ovaries, and female-operated control adult ovaries, showed that
male-grown ovaries lacked vitellogenin, but did internalize other hernolymph
proteins (Fig. 8). The arrowheads in Figure 8 indicate protein subunits which
are found in the male hemolymph and in adult male-incubated ovaries, but
which are not found in pre-vitellogenic ovaries. The three male-grown ovary
samples contain different quantities of protein because the terminal follicles
were of different sizes.
+
DISCUSSION
When ovarian cysts from last instar female larvae were transplanted into
last instar male larvae, follicles detached from the germarium during the larval stage (Fig. 2), increased in size during pharate adult development (Fig. 3),
and formed a chorion with normal surface architecture (Figs. 6, 7). However,
the transplanted ovaries from both males and female-operated controls exhibFig. 6. Scanning electron micrograph of a chorionated egg from a normal adult ovary showing the surface architecture of the chorion. A: Whole view. Arrow 1 indicates the micropile
region and arrow 2 indicates the honeycomb region. Bar length: 100 pm. 6: Enlargement
of the micropile region. Bar length: 10 pm. C: Enlargement of the honeycomb region. Bar
length: 10 pm.
Development of Male-IncubatedOvaries
Fig. 6
229
230
Ballarino et al.
Fig. 7
231
Development of Male-Incubated Ovaries
c
P
C
E"*
0
E
Q
c
6
b
E
P
* -E*
e
I
E
0
0
L
L
C
b
OL
clr
P
0
n
-*
E
Q)
'0
c
E
E
Q
0
E
Q
0
200,
-
116-
97
66
+
43-
Fig. 8. SDS-PAGE analysis of hemolymph and soluble ovary extracts. For each hemolymph
sample, 1 pI of hemolymph was loaded. For each ovary extract sample, except the previtellogenic
ovary, extract from 4 adult eggs was loaded. For the previtellogenic ovary, extract from 4 whole
ovarioles from the 8 day last larval instar was loaded. Molecular weight standards of 200, 116,
97,66, and 43 kD are indicated at the left. Arrows indicate the 2 vitellogenin subunits. Arrowheads
indicate protein subunits which are found in the male hemolymph and in adult male-incubated
ovaries, but which are not found in previtellogenic ovaries. The three male ovary samples contain different quantities of protein because the terminal follicles were of different sizes. std,
standards; op, operated (receivedovary transplant); previtell, previtellogenic.
~
~~
Fig. 7. Scanning electron micrograph ofa chorionated egg from a male-grown adult ovary
showing the surface architecture of the chorion. A: Whole view. Arrow 1 indicatesthe micropile
region and arrow 2 indicates the honeycomb region. Bar length: 100 pm. 6: Enlargement
of the micropile region. Bar length: 10 pm. C: Enlargement of the honeycomb region. Bar
length: 10 km.
232
Ballarino et al.
ited reduced follicle detachment compared to normal control ovaries (Fig. 2),
presumably due to one or more of the following reasons: 1)damage sustained
during transplantation; 2) reduced oxygen supply due to severing of the
tracheoles; 3) loss of anchorage to the body wall leading to reduced contraction of the ovariole wall.
Although the number of follicles that detached from the germarium was
similar in transplanted ovaries from both males and female-operated controls,
the rate of growth during the vitellogenic period of the pupal stage was reduced in male-grown ovaries compared to both female-operated controls and
normal controls (Figs. 3, 4). This reduction in vitellogenic growth may be a
result of 1) the smaller size and lower hemolymph resources of males; and/or
2) the absence of vitellogenin in male hemolymph. A possible role for vitellogenin in protein uptake has been suggested by studies in H . cecropia by
Kulakosky and Telfer [27] in which vitellogenin increased the uptake of other
proteins into follicles incubated in vitro. The absence of vitellogenin in the
male hemolymph may therefore contribute to the reduced vitellogenic growth
seen in male-grown ovaries.
Although the chorion of adult male-grown eggs appeared normal in surface architecture (Figs. 6, 7), choriogenesis was delayed in male-grown ovaries, and a lower percentage of eggs became chorionated (Fig. 5). The process
of chorion formation does not appear to be stimulated by outside hormonal
influences since some ovarioles in male-grown ovaries contained chorionated
eggs and other ovarioles in the same ovary (and presumably exposed to the
same hormonal environment) contained smaller follicles with no evidence
of chorion formation. Was the delay in chorion formation in male-grown
ovaries due to the longer time it took for male-grown oocytes to reach a prescribed size?
Two lines of evidence indicate that oocytes must achieve a prescribed size
before chorion formation is initiated: 1) the small variance in the size of
chorionated eggs within experimental groups; and 2) the presence in adult
male-grown ovaries of ovarioles containing smaller terminal oocytes showing
no evidence of chorion formation, while other ovarioles within the same ovary
contained chorionated eggs of the prescribed size. The cue for the cessation
of uptake and the initiation of chorion formation is unknown and the reason
for the difference in the sizes of chorionated eggs in male-grown, normal, and
female-operated control ovaries is as yet unclear.
SDS-PAGE analysis of hemolymph from males receiving ovaries and of soluble extracts of male-grown ovaries showed that vitellogenin production was
not initiated by the ovary transplant and that only some male hemolymph
proteins were internalized (Fig. 8, arrowheads). Endocytosis of these male
hemolymph proteins thus occurred in the absence of vitellogenin. Zhu et al.
[21] likewise found that male-grown ovaries from B. nzori internalized hemolymph proteins in the absence of vitellogenin. They found that male-grown
ovaries were deficient in vitellin, but contained non-sex-linked 30 kD proteins
found in the hemolymph as well as an egg-specificprotein.
Since endocytosis of male hemolymph proteins occurred in the absence of
vitellogenin, this indicates that the onset of the processes of patency and endocytosis are not cued by the prolonged period of high vitellogenin titers which
Development of Male-IncubatedOvaries
233
are seen in the hemolymph of last instar gypsy moth larvae. Why is there a
prolonged period of high vitellogenin titers in gypsy moths? Perhaps, like storage proteins which are needed during metamorphosis, vitellogenins are produced during the larval feeding stage [23] and simply stored in the hemolymph
in these moths which initiate vitellogenic growth of their oocytes during the
pharate adult stage.
We have shown that ovarian development proceeds normally in males, but
at a slower rate. Why is growth (uptake) slower in males: Is this due to an
unknown hormonal difference between male and female pupae? Or to the absence of the vitellogenin ligand? Or is it simply the result of the smaller size
and lower nutritive resources of male pupae? Studies by Telfer and Rutberg
[19] in H. cecropia showed that male pupae which received ovary implants and
were transfused with hemolymph from female pupae exhibited a greater increase in the size of their oocytes than those which were transfused with male
hemolymph. This indicates that some element in the hemolymph of female
pupae, possibly vitellogenin, enhances oocyte growth. That the responsible
element is vitellogenin is indicated by studies in H. cecropia in which vitellogenin
increased the uptake of other proteins into follicles incubated in vitro [27]. We
will address this question in the gypsy moth in future studies by injecting
purified vitellogenin into male pupae which have received transplanted ovaries, and by incubating follicles in vitro in the presence and absence of vitellogenin and other hemolymph proteins.
Since endocytosis occurs in the absence of vitellogenin, this raises the question of whether vitellogenin-receptorproduction and insertion into membranes
continues in its absence. Another question of interest is whether the accumulative pathway leading to yolk sphere production is altered in the absence of
vitellogenin, as it is in mosquitoes, a species that feeds as an adult [9]. These
questions are answered in part by the following ultrastructural study 1281.
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