/ . Embryol. exp. Morph. Vol. 37, pp. 1-11, 1977
Printed in Great Britain
Binding and uptake of trypan blue by developing
oocytes of Locusta migratoria migratorioides
By ESTHER WAJC, 1 TILLY BAKKER-GRUNWALD 2
AND SHALOM W. APPLEBAUM 1
From the Faculty of Agriculture of The Hebrew University,
Department of Entomology, Rehovot, Israel
SUMMARY
Resorbing oocytes are heavily stained by trypan blue injected into the haemolymph; this
selves as a basis for a quick and convenient method for measuring the degree of resorption.
Oocytes in the beginning of their development are most susceptible towards resorptive
tendencies.
The uptake of trypan blue by normally developing oocytes is proportional to the oocyte
surface. From 'double-marker' experiments, in which trypan blue is injected into the
haemolymph together with [3H]inulin (which does not bind to the oocyte membrane) it is
estimated that the contribution of binding in the interiorization of trypan blue is in the order
of 80 %, under the conditions given.
In vitro incubations show the interaction of trypan blue with the membrane to be electrostatic in nature.
INTRODUCTION
Developing oocytes of the African migratory locust Locusta migratoria
migratorioides pinocytize vitellogenic proteins that are synthesized in the fat
body and transferred in the haemolymph (Tobe & Loughton, 1967; Bentz,
Girardie & Cazal, 1970; Bar-Zev et ah 1975). This uptake mechanism has been
shown to be highly specific in the cecropia moth (Telfer, 1960), other proteins
being barely incorporated. Factors contributing to this specificity may be the
existence of specific receptor sites and/or charge effects on the oocyte plasma
membrane, or the secretion of a follicle cell product which interacts with the
vitellogenins and/or stimulates net pinocytotic uptake (Anderson & Telfer,
1970).
Resorption of developing oocytes may occur until the final stage of vitellogenesis (Tobe & Pratt, 1975), and is influenced by environmental conditions
(Highnam, Lusis & Hill, 1963).
It has been shown (Anderson & Telfer, 1970) that the negatively charged vital
1
Author's address: Department of Entomology, Faculty of Agriculture, The Hebrew
University of Jerusalem, Rehovot 76-100, P.O. Box 12, Israel.
2
Author's address: Department of Physiology, University of Colorado, Medical School,
Denver. Colo. 80220.
2
E. WAJC AND OTHERS
stain trypan blue binds to the membrane of developing oocytes in cecropia, and
is taken up in a way similar to the vitellogenic proteins.
In the following paper, we report on the use of trypan blue in the investigation
of certain quantitative aspects of yolk deposition and its control in Locusta
migratoria migratorioides.
MATERIALS AND METHODS
Locusts were reared under crowded conditions in a continuous light regime.
Females were taken from the cages at various times after the adult moult.
For in vivo experiments, animals were injected with up to 100/d of a 1 % or
1-5 % trypan blue solution (BDH; dialysed against distilled water for 48 h), and/
or with a solution of [3H]inulin (Amersham; specific activity 300mCi/mmol);
1 % or 10 % cold inulin (BDH) was added where indicated (in order to dissolve
this, the solution had to be heated). The animals were left in glass jars or small
cages under illumination (29-31 °C) until haemolymph sampling and dissection
(after 15 min-13 h). At the end of the incubation period, haemolymph samples of
10-20 iA were either added to 2-5 ml 1 % SDS solution for determination of trypan blue content, or to 20 ml counting vials containing 0-2 ml Soluene (Packard)
for determination of radioactivity (see below). Ovaries were removed and
washed twice in locust saline solution (NaCl 170 mM, KC1 6 mM, MgCl2 4 HIM,
CaCl2 2 mM, NaH2PO4 7mM, NaHCO 3 2 mM (Mordue & Goldsworthy, 1969)).
The length and diameter of the oocytes were measured with the aid of
a micrometer scale in the ocular of an Olympus stereoscopic microscope.
Ovarioles were removed from the ovaries, cleaned of the ovarial sheath tissue,
and the terminal oocytes were isolated. Follicle cells were still present at that
stage. Resorbed oocytes (detected as described in 'Results') and fully-grown
oocytes in which 'cap' formation had started were discarded.
The amount of trypan blue associated with the oocytes was determined
essentially according to Anderson & Telfer (1970). For the determination of
[3H]inulin content, 4-20 oocytes (depending upon the size) were solubilized in
1 ml Soluene; 5 ml toluene-based scintillation fluid was added, and the samples
were counted in a Packard Tri-Carb scintillation counter at optimal gain.
For in vitro experiments, the paired ovaries were removed and separated; one
ovary served as a control and was incubated in 1 ml saline solution (NaCl
80 mM, KC110mM, MgCl21 mM,CaCl2 lmM, Na-MES 5 mM, pH 6-5), and the other
in a modified solution, as indicated. After 30min in a shaking bath at 26 °C, the
ovaries were each transferred to 1 ml of the appropriate solutions, but now
containing 0-5 % trypan blue; in some cases, [3H]inulin (2 x 106 cpm) and cold
inulin (1 %) were added. After an additional incubation, samples of the
incubation medium were taken and the ovaries were washed. Trypan blue and
[3H]inulin in the medium and that incorporated in the terminal oocytes were
determined as described for the in vivo experiments.
In all cases less than 10 % of the trypan blue or inulin injected into the
Uptake of trypan blue by locust oocytes
0
1
2
3
4
5
Oocyte length (mm)
6
Fig. 1. Oocyte diameter as a function of oocyte length.
haemolymph (in vivo experiments) or added to the medium (in vitro experiments)
was taken up by the oocytes. Uptake was therefore directly related to the
concentrations of those markers as determined at the end of the incubation
period.
Bovine serum albumin (Fraction V, B grade) came from Calbiochem, dextran
sulphate (M.W. 500000) from Pharmacia, and polyethylene glycol (M.W. 4000)
from BDH.
RESULTS
Dimensions of oocytes
Oocytes have the shape of a prolate spheroid. For each ovary at a given time,
the size of the terminal oocytes is remarkably constant.
Length and diameter of terminal oocytes were measured in ovaries at various
stages of development (Fig. 1). For an oocyte lacking yellow yolk, length is
about 1-4 mm and diameter 0-24mm; just before the detachment from the
ovariole, those values have increased to about 6-4 mm and 1-27 mm, respectively.
During development, the ratio between length and diameter remains approximately five. Thus, measurement of oocyte length suffices for calculation of its
surface and volume (see Appendix, equations 2-3).
Resorption of oocytes during development
After 1 h of in vivo uptake, trypan blue deeply stains oocytes at various stages
of resorption. The stain enters the oocytes and accumulates in vacuoles formed
during the resorption process (Fig. 2A). In normally developing oocytes, in
contrast, the trypan blue staining after 1 h is restricted to a regular pattern of
E. WAJC AND OTHERS
(b)
Fig. 2. Ovarioles containing (A) resorbing terminal oocyte; (B) fully developed
oocyte in which 'cap' formation has started. Oocytes were stained for 1 h in vivo
with trypan blue (see text).
small patches on the oocyte surface (compare Fig. 2B); only after 4 or more
hours is the dye noticeably associated with yolk.
This difference in staining pattern offers a quick and convenient way to
determine the degree of resorption. In Fig. 3 (solid curve), the percentage of
Uptake of trypan blue by locust oocytes
x= 40 -
0
2
4
Oocyte length (mm)
Fig. 3. Resorption as a function of oocyte length. #—ft, Degree of resorption,
determined after 1 h in vivo staining with trypan blue, as described in the text;
, onset of resorption, calculated as derivative of degree of resorption.
oocytes in the process of resorption (calculated as the number of dark-blue
stained oocytes divided by the total number of terminal oocytes x 100) is
plotted as a function of oocyte length: above 4-5 mm, it amounts to more than
40 %. In ovaries with fully developed oocytes (> 6 mm) the absolute number of
darkly stained oocytes decreases (not shown), as many oocytes that have begun
their resorption at an earlier stage are completely resorbed by then. The dotted
curve in Fig. 3 shows the derivative of the solid curve, i.e. the rate of onset of
resorption as a function of oocyte length. Apparently, oocytes at about 3 mm
length (still in the beginning of their development) are under the given conditions most susceptible towards resorption (compare Tobe & Pratt, 1975).
Trypan blue uptake by oocytes in vivo
The amount of trypan blue associated with normally developing oocytes
in vivo was determined as a function of oocyte length at different incubation
times (Fig. 4A). For each size, the trypan blue content increases in time; at
each incubation time, the relation of trypan blue content to oocyte length seems
parabolic. Indeed, plots of trypan blue content against oocyte surface give
straight lines for the different incubation periods (Fig. 4B). In ovaries with
terminal oocytes larger than 6 mm, two extreme states were noticed: oocytes
still attached to the ovariole continued to stain, whilst those found detached in
the oviduct did not bind trypan blue at all; in intermediate cases, 'cap'
formation was clearly visualized (Fig. 2B). This is reminiscent of the 'bipolar'
distribution of vitellogenic protein synthesis found by Bar-Zev et al (1975) and
presumably actuated by cessation of uptake by the terminal oocytes.
Trypan blue presumably binds to the oocyte membrane. Generally, two
terms contribute to total pinocytotic uptake of a substrate: uptake in the liquid
phase and uptake by adsorption on the membrane (Jacques, 1973). As a useful
E. WAJC AND OTHERS
T
80
I
B
A
2
4
Oocyte length (mm)
10
20
Oocyte surface (mm2)
Fig. 4. Trypan blue content as a function of (A) oocyte length and (B) oocyte surface.
Different in vivo incubation times: # , l h ; A, 4 h; • , 13 h. Animals were injected
with 50/A of a solution containing 1 5 % trypan blue.
parameter, the endocytic index has been defined as that volume of external
liquid whose contained substrate is captured by pinocytotic activity (Williams
Kidston, Beck & Lloyd, 1975); thus,
endocytic index = F+Sr
(1)
in which r is the amount of substrate bound per surface area (concentration
dependent, ideally according to a Langmuir adsorption isotherm (Jacques, 1973)),
and F and S are the rates of internalization of external liquid and membrane,
respectively.
In the case of trypan blue, both terms are expected to contribute to uptake.
Thus, the endocytic index will surpass the volume Finteriorized with an amount,
Sr. In order to determine this amount, one should compare the pattern of
uptake of trypan blue with that of a non-binding compound. Inulin, known to be
inert with respect to other membranous systems and neither degraded nor
excreted from the locust haemolymph (unpublished data), was chosen as a
candidate. In separate experiments performed on different days, values found for
uptake of [3H]inulin ranged between 0-7 and 54 nl/mm2/h, but in each
experiment uptake was consistently proportional to the oocyte surface, and
when calculated per surface area, proportional to the incubation time (Table 1);
cold inulin (final concentration in the haemolymph, approximately 0-2 %) did
Uptake of trypan blue by locust oocytes
Table 1. Inulin uptake in vivo
Uptake
XI l^UUdtl^/IJ.
time (h)
2
4
7-5
Cold
inulin
_
+
—
+
—
Oocyte
size (mm)
Surface
(mm2)
60
4-5
3-8
5-2
2-5
3-8
22
12
8-7
16
3-7
8-7
f
nl/oocyte
nl/mm2
nl/mm2/h
80
53
58
130
56
19
3-6
4-4
6-7
81
15
2-2
1-9
2-2
1-8
20
20
0-3
Animals were injected with 10/*1 of a solution containing 6x 105 cpm [3H]inulin, where
indicated preceded by 10 /tl 10 % cold inulin. After the indicated times, haemolymph samples
and oocytes were prepared for scintillation counting as described. Haemolymph volumes
ranged between 500-800/tl.
1
2
3
4
Incubation time (h)
5
Fig. 5. Endocytic index for trypan blue and inulin as a function of incubation time.
# , trypan blue uptake; O, inulin uptake. Animals were injected with 100/tl of
a solution containing 1 % trypan blue, 3 x 105 cpm [3H]inulin and 1 % cold inulin.
not significantly decrease label content. From the fact that the extrapolated
inulin content at zero time is zero, and that cold inulin in the given concentration does not act as a competitor, it can be inferred that inulin is inert with
respect to oocyte membranes too, and that its endocytic index at any moment
represents the real volume of fluid interiorized.
In Fig. 5, the compiled results of some experiments are shown in which trypan
blue and [3H]inulin were injected simultaneously. A certain amount of trypan
blue (corresponding to 4 nl/mm2) appears to be associated with the oocytes at
zero time, and the increase in trypan blue content is about six times as fast as
that in inulin content. Values for inulin uptake were rather scattered, but in
E. WAJC AND OTHERS
Table 2. Inulin uptake in vitro
Uptake
Addition
30 % haemolymph
Incubation Oocyte
time (h) size (mm)
I)
I)
Surface
(mm2)
3-8
8-7
3-5
7-3
nl/mm2
nl/oocyte
12-1
\40
|2-1
11-9
0-3
0-5
0-3
0-3
Two pairs of ovaries were incubated in 1 ml standard saline or saline supplemented with
30 % haemolymph taken from the females used for the experiment. 2 x 106 cpm [3H]inulin
was added. Of each pair, one ovary was taken out after 2 h, the other after 4 h and, together
with 10 /tl samples of the incubation medium, prepared for scintillation counting.
Table 3. Factors influencing in vitro binding of trypan blue
Binding (n moles/cm2)
Modification of standard conditions
pH = 7-5
-Mg2+, -Ca2+, +lmMEDTA
Serum albumin (5 mg)
Dextran sulphate (10 mg)
Polyethylene glycol (10mg)
Control
Experimental
% effect
2-8
3-7
3-2
38
27
1-6
20
2-3
3'1
2-6
-43
-46
-27
-17
-3
The two ovaries of one pair were separately incubated for 0-5 h in 1 ml standard saline
or modified saline, both containing 0-5 % trypan blue.
a separate experiment (not shown) it was established that uptake was depressed
compared to that in the absence of trypan blue; inhibition ranged between
40 and 80 %
In vitro binding of trypan blue
The amount of trypan blue associated with terminal oocytes after in vitro
incubation, as in the in vivo experiments, increases proportionally to the oocyte
surface (not shown), allowing us to express the trypan blue content in units per
surface area in these experiments too.
Oocytes in vitro under the given conditions take up only very small amounts
of [3H]inulin; haemolymph does not stimulate, and uptake is virtually finished
within 2 h (Table 2). In the presence of 0-5 % trypan blue, values found are even
lower (not shown). This means that most (as can be calculated, more than 90 %)
of the trypan blue is externally bound. Thus, the in vitro system should be an
appropriate model for the investigation of factors influencing binding.
As can be seen (Table 3), trypan blue binding decreases with external pH and
is Mg 2+ and Ca 2+ dependent; negatively charged macromolecules like serum
Uptake of trypan blue by locust oocytes
9
albumin (probably as a serum albumin-trypan blue complex (Lloyd, Beck,
Griffiths & Parry, 1968) and dextran sulphate act as competitors, while
neutrally charged polyethylene glycol has no significant effect.
DISCUSSION
In our experience, trypan blue proved to be a quick and convenient indicator
of resorbing oocytes, which could prove useful in other studies concerned with
resorption.
Physiologically, trypan blue has toxic properties (Lloyd et al. 1968); as shown
for the cecropia moth (Anderson & Telfer, 1970) and especially relevant here, it
inhibits pinocytotic uptake of vitellogenic proteins. In our experiments, this
expresses itself in the fact that trypan blue suppresses the uptake of inulin, i.e. net
volume uptake. The reason for this may be either that vitellogenic proteins are
essential for stimulation of pinocytosis and that trypan blue competes with them
for access to the membrane, or that trypan blue in itself inhibits pinocytosis.
Thus, trypan blue is not the best model compound one could choose for
vitellogenin uptake studies. However, the fact that its uptake by the oocytes is
proportional to the oocyte surface (Fig. 4B) is of general importance: it suggests
that this may apply to vitellogenic proteins as well, which means that throughout development the pinocytotic activity per surface area would be constant,
and volume increase would be proportional to the oocyte surface. (It should be
realized that equation 2, Appendix, gives a value for the enveloping surface, and
that the actual surface involved in pinocytosis is many-fold larger, due to the
presence of microvilli; however, it seems reasonable to assume that the ratio
between actual and enveloping surface area throughout development is constant.)
In fact, via elementary calculus this can already be inferred to be the case from
the experimental finding (see for instance Highnam & Haskell, 1964, Fig. 1,
curve for flown-crowded animals, and Tobe & Pratt, 1975) that oocyte length
increases approximately linearly in time; derivation is given in Appendix,
equations 4-5. After insertion of the appropriate constants it can be calculated
that oocytes, in order to complete their development from 1-5 mm length till
6-5 mm length in 7 days, should continuously interiorize about 5 nl/mm2
surface/h (Appendix, equation 6), and this is indeed the maximal value calculated
from [3H]inulin uptake. This again suggests that the endocytic index for [3H]inuJin represents the real volume interiorized. Other labelled compounds have
been used for quantitative determination of pinocytosis, such as [3H]sucrose
(Wagner, Rosenberg & Estensen, 1971) and [125I]polyvinylpyrrolidone (Williams
et al. 1975), but for our purposes [3H]inulin seems to be preferable because,
contrary to sucrose, it is not metabolized in the locust haemolymph, and
eventually it can be used in double-labelling experiments together with
a [14C-]isotope.
The difference in slope in the trypan blue curve and the inulin curve in Fig. 5
10
E. WAJC AND OTHERS
indicates how much faster trypan blue is taken up than inulin; it also gives the
concentration factor for trypan blue. As the difference in slope is approximately
a factor of 6, the binding contribution in the uptake of trypan blue must be
about 5/6 = 80 % (compare equation 1). In principle, this contribution depends
upon the amount r bound per surface area, and thus upon the concentration of
trypan blue and the presence of competitors (such as vitellogenins).
The trypan blue content at zero time represents strong binding, both to the
plasma membrane (functional in pinocytosis) and at other places not relevant for
uptake (for instance, in between the follicle cells) (Anderson & Telfer, 1970).
Probably, during pinocytosis an additional number of weak binding sites are
occupied, that lose their bound dye molecules upon washing.
From the in vitro experiments (Table 3), it appears that trypan blue is bound
by virtue of its negative charge: cations such as Mg 2+ , Ca 2+ and H+ enhance
binding, whilst poly-anions act as competitors (compare also Wallace & Ti,
1972). Although our in vivo data show that in principle those electrostatic
effects could play a role in selective uptake, it should be realized that vitellogenins
relatively are much stronger concentrated in the oocyte yolk than trypan blue;
if, like in the cecropia moth (Telfer, 1960), the concentration factor is 20, the
uptake of vitellogenins should be 20 x as fast as that of a non-binding compound, which means that the binding contribution should amount to 19/20
= 95°/
yj
/
0
.
Thus, in the binding and uptake of natural vitellogenins factors other than
electrostatic ones might contribute, such as the presence of specific receptors or
the secretion of follicle cell product previously mentioned (Anderson & Telfer,
1970). These factors will be evaluated with vitellogenic proteins which are
presently being isolated and radio-labelled.
This work was supported in part by a grant from the Volkswagenstiftung, Hannover.
Drs Esther Wajc and Tilly Bakker-Grunwald were Lady Davis Fellows of The Hebrew
University of Jerusalem.
APPENDIX
Oocytes have the form of a prolate spheroid, with length / = 5 times
diameter d (Fig. 1). Thus,
and
Surface, s = n.d.l = 0-62/ 2
(2)
volume, v = n {£)* (l ~ f) = 0-03 /3
(3)
Experimentally (Jaques, 1973; Wagner et al. 1971) it is found that in first
approximation oocyte length increases linearly in time, from 1-5 mm till
6-5 mm in 7 days. Thus,
-T = constant = 0-03 mm/h.
(4)
Uptake of trypan blue by locust oocytes
11
The volume increase in time is:
^
= ^ . ^
= 3.0-03 /2.0-03 mm/h = 0-003 / 2 mm3/h (1 in mm),
(5)
which is seen to be proportional to the surface.
The volume increase in time per surface area can be calculated as:
dv 1
0-003/ 3
_ A r . c ,,
„,,
. ,
„,,
—.- =
= 0-005 /d/mm2/h = 5nl mm2/h,
at s
U-o 1
,,.
(6)
Compensatory water movements other than pinocytotic volume uptake, that
could occur because of osmotic effects during the phase transition of soluble
vitellogenins to dispersed yolk proteins, are not taken into consideration.
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