/ . Embryo!, exp. Morph. Vol. 40, pp. 187-197, 1977
Printed in Great Britain © Company of Biologists Limited 1977
On the possibility of sperm aster
involvement in dorso-ventral polarization and
pronuclear migration in the amphibian egg
By MARIO E. MANES1 AND FRANCISCO D. BARBIERI1
From the Institute de Biologia, Universidad National de
Tucumdn, Argentina
SUMMARY
Sperm aster involvement in the determination of the plane of bilateral symmetry of the
future embryo as well as pronuclear movement and orientation has been investigated in the
amphibian egg. The procedure involved injecting very small amounts of disrupted sperm
cells into the subcortical region of the egg of the toad Bufo arenarum. Using this procedure
we have previously shown that structural integrity of the sperm cell is not required for the
determination of the symmetry plane.
The present report deals with the cytological effects induced by the injection of sperm
homogenate into the egg. As expected, an aster was always present in these eggs. This aster
closely resembled that produced in the fertilized egg as the sperm nucleus migrates through
the egg mass.
Sections of injected eggs have also shown the presence of two pigment trails similar in
appearance to those produced by pronuclear movement after normal fertilization. The
streak analogous to the sperm entrance track emerged from the point where the pipette was
introduced.
None of these effects were observed in Ringer's injected eggs. At the time of the rotation
of symmetrization, the female pronucleus was seen within a clear area of cytoplasm located
at a short distance below the egg cortex. An aster field organized around this clear area was
probably formed as a result of the egg centriole activation. In no case was the egg nucleus
seen to migrate towards the copulation site as was seen to occur in sperm homogenate-treated
eggs.
The active agent was found to be a particulate component which sediments with the head
but not the tail fraction of Bufo arenarum sperm.
Injection of blood cells caused a similar response to that elicited by the sperm homogenate.
INTRODUCTION
The amphibian egg presents outwardly an animal region heavily pigmented
and a vegetal region with little pigment. The centre of both of these hemispheres
define a polar axis around which cytoplasmic components are evenly distributed.
As the result of a structural rearrangement operating fairly soon after fertilization, this radially symmetrical pattern is lost and the dorso-ventral polarization
1
Authors' address: Instituto de Biologia, Chacabuco 461,4000 San Miguel de Tucuman,
R. Argentina.
188
M. E. MANES AND F. D. BARBIERI
of the future embryo is definitely established. The first external sign of this
alteration is the appearance of the grey crescent, lying at one side of the egg
between the animal and the vegetal regions. At this stage, the plane of bilateral
symmetry of the future embryo is defined by the polar axis of the egg and the
centre of the grey crescent, which marks the dorsal side of the future embryo
(Ancel & Vintemberger, 1948; Pasteels, 1951). Experiments carried out in
different species indicate that the spermatozoon plays an important role in the
determination of the plane of bilateral symmetry and, hence, in the location of
the grey crescent, which generally appears at the opposite side to the point of
sperm penetration (Ancel & Vintemberger, 1948; Holtfreter & Hamburger,
1955; Clavert, 1962; Pasteels, 1964; Lovtrup, 1965). As a first approach to the
investigation of the mechanism underlying this effect, experiments were designed
to determine whether sperm cell integrity is required to produce egg polarization. This work showed that as a result of the subcortical injection of sperm
homogenate into the egg of the toad Bufo arenarum, the grey crescent is always
formed at the side opposite to that of the injection point (Manes & Barbieri,
1976). To explain this result, we suggested that it was produced by an aster
which in turn was organized by the material injected into the egg. This hypothesis was based on some observations reported by Kubota (1967), who noted
that dorso-ventral polarization of the egg coincides in time with a rigidity
change in the cortex, which appears always to be underlain by the sperm aster
or a cytaster.
The sperm aster has also been considered as a good candidate to account for
the migration of the male pronucleus toward the centre of the egg (Kostanecki
& Wierzejski, 1896, according to Brachet, 1910; Herlant, 1911, 1913). In this
interpretation, the nucleus is assumed to be passively pulled along after the
aster as it moves actively toward the centre of the egg as a result of the tendency
of its radiations to occupy most of the available extension of yolk-poor cytoplasm. More recently, Subtelny & Bradt (1963) have reported that blastula
nuclei transplanted into Rana pipiens egg migrate through the egg mass forming
a pigmented trail in a similar manner to that formed by the sperm nucleus. The
authors assume that the movement of the injected nucleus may be accomplished,
at least in part, by the growth of the astral system organized around it, a possibility which they consider accessible to experimental attack. No such, experimental analysis for the amphibian egg seems to have been reported up to the
present, however. As far as we know, experimental substantiation of sperm
aster involvement in the migration of the sperm nucleus has been obtained for
the sea urchin only, in work with eggs fertilized in glass capillaries of small
diameter (Allen, 1954).
With these issues in mind we have extended our microinjection experiments
using the Bufo arenarum egg system for three purposes. First, to establish
whether the sperm homogenate which proved to be an effective polarityinducing agent is also effective at inducing aster formation. Second, to test
Pronuclear migration in amphibian eggs
189
whether an aster artificially induced under the egg cortex has the capacity to
migrate through the egg mass pulling along pigment granules as the sperm does
in normal fertilization. Third, to assay some sperm fractions for their ability
to induce the formation of asters and pigmented streaks, as well as egg
polarization.
MATERIALS AND METHODS
The material used was the toad Bufo arenarum, collected in the vicinity of
Famailla and stored at 15 °C. Mature eggs, obtained 12 h after injection of
homoplastic hypophysis suspension, were removed from the ovisacs.
Washed spermatozoa were ruptured by two passages through a French pressure cell at 2 x 108 N.m~ 2 and injected into the unfertilized egg according to the
procedure detailed previously (Manes & Barbieri, 1976). In some experiments
the particulate fraction of the homogenate was separated by centrifugation at
50000 g for 60 min. The resulting supernatant and the pellet, resuspended in
10 % Ringer solution without bicarbonate, were then separately injected into
different eggs. Similar experiments were carried out with head and tail fractions
of Bufo arenarum sperm prepared by the procedure of Mailer et al. (1976).
Blood cells of the same species have been also assayed by microinjection into
the egg of the toad. Blood was drained from pithed animals into three vols of
10 % Ringer solution with added sodium citrate, and stored in an ice bath
during injections. In some experiments, blood cells, suspended in three vols of
10 % Ringer solution, were ground in a mortar with crystalline alumina 90-mesh
(Norton Co.) prior to injection. The volume of material injected into each egg
was below 5 nl. In some experiments, electrolytical marks (Ancel & Vintemberger, 1948) were used to facilitate the detection of the injection point. In order
to get reproducible results, an electrical biological stimulator was employed
(Carlino, Valentinuzzi, Torres & Arredondo, 1974).1 The egg was placed on a
silver plate over a small piece of black paper soaked with 10 % Ringer solution.
The plate was connected to the negative terminal of the generator while the
platinum marking needle was connected to the positive terminal. Good results
were obtained with single pulses of 20 V in amplitude and 0-1 msec duration.
Four lots of control eggs were used. One was injected with Ringer solution
under the same conditions as experimental eggs. Other control eggs were
covered with (a) an aliquot of the same sperm suspension used to prepare the
homogenate, (b) sperm homogenate and (c) 10 % Ringer solution.
Eggs were fixed about 90 min after injection, when direct observation showed
that the rotation of symmetrization was accomplished. As an easy procedure
to detect the presence of pigment streaks, eggs were directly dissected under the
stereomicroscope after fixation in 6 % glutaraldehyde in 0-1 M phosphate buffer
1
This instrument was developed with the support of the Secretaria de Ciencia y Tecnica,
Universidad Nacional de Tucuman, to the joint Bioengineering Program between the Instituto
de Ingerieria Electrica and the Instituto de Biologia.
13
EMB 40
190
M. E. MANES AND F. D. BARBIERI
Table 1. Induction of pigment streak in sperm homogenate injected eggs
All data are expressed as percentages (mean and standard error from
six experiments carried out with different animals).
Treatments
Injected eggs
Sperm homogenate
Ringer solution
Control eggs
Sperm suspension
Sperm homogenate
Ringer solution
Streak
induction
Fertilized
eggs
Activated
eggs
97
103
90-7 ± 3 0
0
—
—
—
—
1292
1540
1532
—
—
—
991 ±0-5
01 ± 0 1
0
10 ±0-5
0-8 ±0-4
1-2 ±0-5
No. of
eggs
(pH 7-6). For cytological examination, eggs were fixed in Smith's solution and
sections, after being routinely prepared, were stained either in Ehrlich's hematoxylin and eosin or light green. Some preparations were also mounted unstained to observe changes in the pigment distribution. Cytological sections
examined include 52 injected eggs, 22 unfertilized eggs and 47 fertilized eggs
fixed at times after insemination.
RESULTS
The present results have confirmed that in sperm homogenate-injected eggs
the grey crescent is consistently formed at the opposite side to the point where
the microneedle was introduced (Manes & Barbieri, 1976). In addition, the
experiments here reported show that the determination of the plane of bilateral
symmetry is always accompanied by aster formation and the establishment of
pigmented streaks which resemble those produced by the pronuclei as they
progress inside the egg. Table 1 shows the percentage of injected eggs in which
this latter effect was observed. That these results were not due to the presence
of contaminating whole sperm in the material injected into the egg is indicated
by the low fertilizability of control eggs covered with an aliquot of the sperm
homogenate.
The following results consist of a cytological analysis of sperm homogenateinjected eggs as compared to those injected with Ringer solution. This will be
complemented by the description of microinjection experiments designed to
assay the particulate and nonparticulate fractions of the homogenate, as well
as blood cells, for their ability to induce similar effects.
Ringer's injected eggs
All control eggs injected with Ringer saline exhibited typical signs of activation, such as extrusion of the second polocyte, rotation of orientation and the
appearance of the grey crescent. In contrast to sperm homogenate-injected eggs,
Pronuclear migration in amphibian eggs
191
Fig. 1. Section of an egg fixed 90 min after injection with Ringer solution. Note a
clear area of cytoplasm surrounded by an ample aster field (a) containing the
female pronucleus (arrow) (b). x 165 and x 625.
Fig. 2. Comparison of the pigment trail emerging from the injection point of a
sperm homogenate-injected egg (a) with the sperm track of a fertilized egg (b).
Although difficult to appreciate in (a), a wide non-filamentous aster area is organized around the artificially induced streak. Both eggs were fixed as soon as the
rotation of symmetrization was accomplished, x 160.
Fig. 3. Photomicrographs of unstained sections. Note the pigment streak (/)
marking the displacement of the female pronucleus in a sperm homogenate-injected
egg (a) and a fertilized egg (b). t, pigment trail emerging from the injection point;
s, sperm track, x 160.
13-2
192
M. E. MANES AND F. D. BARBIERI
no relationship was observed between the region where the crescent was formed
and the point where the microneedle was introduced.
Sections of these eggs showed that the female pronucleus formed at the completion of meiosis descended a short way following the polar axis of the egg.
This was indicated by a pigment track extending from the animal pit, to a welldefined area of clear cytoplasm where the pronucleus could be visualized with
relative ease (Figs. 1 a, b). The clear area, shaped like a slightly depressed
sphere, appears delimited by a denser pigmented area. The cytoplasm is organized there as a loose network formed by trabeculae containing small yolk platelets and pigment granules. Another constant feature of Ringer-injected eggs was
the presence of an ample aster around this clear area. Although astral filaments
were not discernible, yolk platelets and pigment granules appeared radially
arranged so as to form rows separated by thin spaces of cytoplasm. The astral
region was lightly stained and poorly pigmented, whereas its margins appeared
often deeply stained and denser in pigment.
Division of the aster or the female pronucleus was never observed in Ringerinjected eggs.
Sperm homogenate injected eggs
Observation of sectioned eggs showed that the injection of sperm homogenate
caused a response pattern different from that obtained by the injection of
Ringer solution. The most striking feature was the formation of the pigment
trail akin to the sperm entrance track. This artificially induced entrance path
was always found to originate from the entrance point of the pipette tip.
Detailed cytological examination showed that it does not differ at all from the
sperm track as observed in the normally fertilized egg (Figs. 2a, b). Around
the injection point, pigment granules from the egg cortex flow in forming a dark
cone with its vertex pointing toward the centre of the egg and from which a line
of pigment continues through the egg substance to reach the presumptive dorsal
region. The cytoplasm around this streak always showed a radial non-filamentous arrangement occupying most of the volume of the animal hemisphere
(Fig. 2a). This astral formation was by no means comparable to that found in
fertilized control eggs fixed at the same time, where the first mitotic division
was already in telophase and the asters, at the poles of the spindle, were filamentous and smaller in size (Fig. 2b). The large astral region of homogenateinjected eggs resembled the aster formed around the sperm nucleus at an
earlier stage as it approaches the site of nuclear copulation. Except for a single
specimen, where multiple ill-defined aster areas were seen, a unique aster per
egg was consistently observed. No structure that could be regarded as a nucleus
was ever observed at the centre of the aster field of these eggs.
Another important difference between eggs injected with Ringer solution and
with sperm homogenate concerns the behaviour of the female pronucleus and
its corresponding centrosome. Even though it was not possible to locate the
Pronuclear migration in amphibian eggs
193
Table 2. Streak-forming response of eggs to different injected sperm fractions
Treatments
Injected eggs
50000 g pellet
50000 g supernate
Head fraction
Tail fraction
Control eggs*
Sperm suspension
Sperm homogenate
Ringer solution
No. of
eggs
42*
45*
20f
20f
974
1056
962
Streak
induction
Fertilized
eggs
Activated
eggs
1000 ±0
4-3 ±2-5
—
—
—
—
—
—
—
—
98-5 ±0-8
01 ± 0 1
l-5±0-8
l-7±l-2
20±l-7
550
0
—
—
—
0
Legend as in Table 1.
* N = A.
t N=l.
female pronucleus in these eggs, its migration was clearly indicated in unstained
sections by a streak of pigment. The pronucleus descended a short gap along the
polar axis of the egg, but instead of stopping there as in Ringer-injected eggs, it
veered away in the same direction as the pigment trail emerging from the
injection point. As in the normally fertilized egg, the centriole associated with
the egg nucleus showed no sign of activity (Figs. 3 a, b).
At the time the grey crescent was formed, no amphiaster nor spindle could
be observed.
Association of activity with paniculate sperm components
We will now consider the results of some experiments in which the capacity
of Bufo arenarum sperm fractions to induce the effects above described has been
tested. Due to the difficulty of obtaining pure preparations of sperm fractions,
we have not yet been able to determine the cell component responsible for those
effects. We do have evidence, however, that activity to induce aster and streak
formation, and egg polarization, is linked to some particulate sperm component,
and that sperm flagella are completely ineffective at inducing such effects. Thus,
the data shown in Table 2 demonstrate that upon centrifugation at 50000 £
for 60 min, streak-inducing activity sediments with the pellet. As shown in the
same table, when equal amounts of sperm head and tail fractions were injected
into different eggs, only those treated with the head fraction exhibited the
presence of pigment trails. Under the microscope most heads exhibited a short
stub of the flagellum, and the tail fraction appeared free of any visible contaminating material.
The changes induced by injection of these materials into the egg were indistinguishable from those determined by injection of the whole homogenate.
194
M. E. MANES AND F. D. BARBIERI
Table 3. Streak-forming response of eggs to injected blood
Treatments
Injected eggs
Whole cells*
Disrupted cellsf
Control eggs*
Sperm suspension
Sperm homogenate
Ringer solution
No. of
eggs
35
18
Streak
induction
Fertilized
eggs
Activated
eggs
98-5111
0
0
1-5 i l l
1-7 + 1-7
2-312-3
16-713-3
7001301
686
742
962
Legend as in Table 1.
* N = 3.
t N=2.
Blood cells
In order to establish whether the response obtained was due to some specific
sperm component, we decided to assay blood cells of the same species. These
cells were chosen because of their well known property of acting as a co-factor
in inciting the amphibian egg to undergo parthenogenetic development (Bataillon, 1911, 1929; Bogucki, 1921, according to Shaver, 1953). As shown by midsections of glutaraldehyde-fixed eggs, blood cells were effective at inducing
typical pigment streaks (Table 3). The inspection of cytological sections of these
eggs showed the presence of an asterareaof the type found in sperm homogenateinjected eggs.
DISCUSSION
The results described here lend further support to the hypothesis that dorsoventral polarization of the amphibian egg is mediated by aster formation
(Kubota, 1967; Manes & Barbieri, 1976). Thus, an aster was always present in
sections from sperm homogenate-injected eggs and the active fraction probably
contains centrioles as the effective agent as discussed below.
Trails of pigment granules similar to those produced as the pronuclei migrate
from the cortex were always observed along with asters in sperm homogenateinjected eggs. The fact that an artificial sperm track can be only induced as far
as an aster is formed suggests that pigment granules are pulled along after the
aster as it displaces through the egg mass. This strongly supports the contention
then that in normal fertilization the male pronucleus is also passively pulled
along as the aster moves actively toward the centre of the egg.
It would be of utmost interest to establish whether a causal relationship does
also exist between sperm aster formation and the movement and directionality
of the female pronucleus. In Ringer-injected eggs the female pronucleus descended following the egg axis, but in no case was it seen to move dorsad as was
Pronuclear migration in amphibian eggs
195
always found in fertilized or sperm homogenate-injected eggs. The cytological
response elicited in these control eggs was essentially the same as that reported
for the artificially activated egg of Rana fusca (Herlant, 1913). A further point
is that, if it is true that the male pronucleus movement is caused by the growing
fibres of the sperm aster, it could equally be argued that the descent of the
female pronucleus in the egg pricked with a clean glass needle is determined by
the growth of its associated aster below the animal pole of the egg.
The response elicited in the egg by microinjection of blood cells was analogous
to that produced by injection of sperm homogenate. A puzzling question is why
the effects here described were not observed in the egg of Rana fusca when
activated by pricking in the presence of blood. According to Herlant's (1913)
report, this egg displays an active centre of its own concomitantly with cytasters
formation around the point where the needle was inserted, but no reference is
made about the presence of pigment streaks. Despite the lack of information to
account for this difference, we believe that it can be explained more in terms of
the activation procedure applied or the number of eggs examined, than of the
species used.
If it is true that pronuclear movement and directionality as well as egg polarization depend on aster formation, the prime mover of all these events is likely
to be the centriole provided by the sperm. This conclusion is based on the
following observations: (1) the active component is a particulate sperm structure; (2) it is associated to the sperm head but not the tail; (3) the only sperm
fraction capable of inducing spindle formation in the frog egg was reported to
be the centriole-containing fraction (Mailer et al. 1976); and (4) evidence exists
indicating that materials from other cells effective at inducing asters in amphibian eggs are also likely to contain centrioles as the active agent (Heidemann &
Kirschner, 1975). Since several centrioles were probably introduced into each
egg, the question arises as to why a single aster field was generally formed. This
might be tentatively explained by assuming that the first injected centriole which
becomes active, exerts a suppressing influence on the remaining centrosomes
(Herlant, 1911). It is interesting to note in this respect that the results of the
present study provide additional support for the view that the sperm aster also
exerts a suppressing influence on the centriole associated to the female pronucleus (see Briggs & King, 1959). This is indicated by the fact that the egg
centriole becomes activated in Ringer-injected eggs but not so in fertilized or
sperm homogenate-treated eggs.
The present investigation showed that microinjection of a very small amount
of sperm homogenate into the subcortical region of the amphibian egg provides
the possibility of inducing the whole process of egg activation excepting nuclear
association, together with the possibility of fixing at will the location of the
grey crescent and, thence, of the plane of bilateral symmetry of the future
embryo. This should prove to be a useful tool for the experimental analysis of
certain aspects of egg activation.
196
M. E. MANES AND F. D. BARBIERI
It is certainly true that as in Boveri's days, when the essential event of fertilization was believed to be the introduction of the sperm centriole into the
egg, this cell organelle is exciting to us again (Mazzia, 1961).
Appreciation is expressed to Mr Eduardo Rothe for his assistance in the preparation of
the manuscript. This work was supported in part by grants from the Consejo Nacional de
Investigaciones Cientificas y Tecnicas (R. Argentina) (CONICET), The Population Council,
New York, N.Y. (awarded to the CONICET) and the Fundacion Lucio Cherny (R. Argentina). M.E. M. was supported by a scholarship from the CONICET.
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