/ . Embryol. exp. Morph. Vol. 32, 2, pp. 287-295, 1974
Printed in Great Britain
287
Biochemical control of fresh-water
sponge development : effect on DNA, RNA
and protein synthesis of an inhibitor
secreted by the sponge
ByFRANCINE ROZENFELD 1
Laboratoire de Biologie animale et cellulaire, Université libre de Bruxelles
SUMMARY
During their growth, fresh-water sponges release into the medium a dialysable inhibitor
(gemmulostasin) which reversibly blocks development of the gemmules at an early phase, prior
to the first mitoses.
The incorporation of [3H]labelled precursors into DNA, RNA and proteins has been
measured throughout development, in the presence or absence of inhibitor. Autoradiographic
controls of the nuclear incorporation of [3H]thymidine have been made. Gemmulostasin
erases the peak of [3H]thymidine incorporation that otherwise occurs just before the first
mitoses. Its overall effect is to bring about, either directly or indirectly, a high peak in the
incorporation of [3H]uridine and [3H]leucine at a time when the incorporation of these
precursors is low in the controls.
It is suggested that a causal relation exists between these phenomena.
INTRODUCTION
In temperate climates the fresh-water sponges depend on their gemmules to
withstand the frosts of the winter. It is therefore essential to them that their
gemmules be kept in dormancy from the moment they are formed, during the
summer, until the next spring. While in some species this goal is achieved by
gemmular diapause (Rasmont, 1954a, b; 1955), in Ephydatia fluviatilis the living
sponge inhibits the development of its gemmules by means of a diffusing
dialysable inhibitor, gemmulostasin (Rasmont, 1963,1965), the chemical nature
of which is still unknown.
It has been established that this agent reversibly inhibits the development of
gemmules at an early stage, prior to the first mitoses, and that it is potent in
inhibiting the development of gemmules of diapausing as well as of nondiapausing species (Rozenfeld, 1970). It is therefore likely that this inhibitor
acts on a fundamental mechanism preparatory to cell division.
1
Author's address: Laboratoire de Biologie animale et cellulaire, Université libre de
Bruxelles, 50 av. F. D. Roosevelt, 1050 Bruxelles, Belgium.
19
E M B 32
288
F. ROZENFELD
In the present work, we have investigated the biochemical events (DNA, RNA
and protein syntheses) which take place during that particular stage of development, and the way in which the inhibitor interferes with their normal course.
MATERIAL AND METHODS
Biological material
The gemmules of Ephydatia fluviatilis (strain a) (VanDeVyver, 1970) were
gathered, in the autumn, from open-air cultures in a pond near Brussels. They
were kept at 0 °C until the experiments were performed. Before use, they were
superficially sterilized with dilute hydrogen peroxide. Unless otherwise specified,
the incubation medium was mineral ' M ' medium (Rasmont, 1961).
For the production of gemmulostasin, we used populations of sponges
hatched from 200 gemmules, cultivated for 15 days in 25 ml Petri dishes, in
constant darkness at 20 °C. The resulting conditioned medium was concentrated
by vacuum evaporation and assayed for gemmulostasin activity according to a
previously described technique (Rasmont, 1965).
Biochemical analyses
Batches of 1000 gemmules were incubated in 5 ml of the mineral medium (M)
or in 5 ml of the conditioned medium with an inhibitory activity of 20 units (G).
Every 6 h during the incubation, three batches of gemmules were removed
from the mineral medium, three other batches from the conditioned medium and
all of them transferred to fresh medium (either mineral or conditioned) containing the radioactive precursor.
After a 12 h pulse the gemmules were transferred to a medium containing
non-radioactive precursor, and washed three times.
Three parallel experiments were conducted along these lines. In one experiment
the precursor was [3H]thymidine (10 /^Ci/ml, specific activity 20-6/*Ci/mg), in the
second it was [3H]uridine (10/^Ci/ml, specific activity 18-6ytóCi/mg), in the
third it was [3H]leucine (10 /^Ci/ml, specific activity 3-8 /^Ci/mg) (labelled precursors from the Radiochemical Centre, Amersham, England).
After having been washed in non-radioactive medium, the gemmules were
gently crushed in cold double-distilled water and the cells separated from the
empty shells by sedimentation. The cells were homogenized in a glass homogenizer and by alternate freezing and thawing. A microscopic examination
revealed very few unbroken cells.
DNA, RNA and proteins were selectively extracted with trichloracetic acid,
according to the Kennel procedure (Kennel, 1967). However, because a specific
precursor had been selected for each class of macromolecules, it was not necessary to purify each fraction. Therefore, we did not use filters to separate the
radioactive precipitable material from the non-radioactive soluble material.
The amount of radioactivity incorporated into each sample was measured in a
Biochemical control of sponge development
289
Nuclear Chicago liquid scintillation counter ; the final result for each sample was
expressed in disintegrations per minute (dpm) per gemmule. The recorded
countings were corrected for quenching and background.
The whole experimental procedure, from incubation until scintillation counting, was performed three times for each precursor. The results of the triplicate
experiments were quite parallel.
In Figs. 1, 4 and 5 the three points given for each time value relate to the three
parallel batches of the third experiment.
Autoradiographic studies
Batches of 15 gemmules were incubated either in 1 ml of the mineral medium
or in 1 ml of the conditioned medium (inhibitor activity : 3 units) each containing
[3H]thymidine (20 /id), [3H]uridine (20 /*Ci) or [3H]leucine (20 ^Ci).
Every 12 h, treated and control batches were removed, washed 3 times with
non-radioactive precursors, fixed with glutaraldehyde, dehydrated, embedded in
Paramat (Gurr) and sectioned at 5 jam.
Depending on the precursor used, the sections were treated either with a solution of deoxyribonuclease (Worthington : 0-2mg/ml in Tris buffer at pH 7-5
containing MgCl2 M/300) for \\ h at 37 °C or with a solution of ribonuclease
(Sigma: 0-2 mg/ml in Tris buffer). Control sections were treated with the buffer
only.
The slides were then transferred to 5 % trichloracetic acid at 4 °C for 5 min
to remove unincorporated nucleotides, and washed in running water for 30 min.
All sections were finally dipped in Ilford K2 emulsion and kept for 3 weeks at
4 °C (Kopriwa & Leblond, 1962). After development the preparations were
stained with methyl green-pyronin (Brächet, 1941) and mounted.
RESULTS
Effects of gemmulostasin upon the incorporation of labelled thymidine into DNA
The radioactivity of DNA isolated from gemmules incubated either in
mineral medium (M) or in conditioned medium (G) is plotted as a function of
incubation time in Fig. 1.
As can be seen, the uptake of labelled thymidine during the normal development of the gemmules (curve M) remains very low throughout the first 30 h of
the incubation. It then increases markedly during the next few hours, reaching its
maximal value at 36 h of incubation.
During subsequent hours, the DNA radioactivity appreciably decreases, but
is still high until the day before hatching.
As can be seen from the autoradiography, [3H]thymidine was found to be
incorporated into the nuclei of gemmules fixed at and after the 24th hour of
incubation (Figs 2, 3). It should be kept in mind that the first mitoses occur
between 36 and 48 h of incubation (Rozenfeld, 1970).
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Fig. 1. Radioactivity of DNA extracts from gemmules during their incubation in
mineral medium ( # , M) and in conditioned medium (O, G), after a 12 h pulse.
Groups of 3 points represent the data for the 3 parallel batches of one experiment.
In the conditioned medium (Fig. 1, curve G), the uptake of labelled thymidine
is persistently inhibited to the level attained in the first 30 h in the controls.
In parallel conditions, no labelling of the nuclei could be detected by autoradiography.
This work thus leads to the conclusion that an overall effect of gemmulostasin
is an inhibition of the nuclear incorporation of thymidine.
Effects of gemmulostasin upon the incorporation of labelled leucine into proteins
Figure 4 shows the rate of incorporation of [3H]leucine into the proteins of
gemmules, either in mineral medium (M) or in gemmulostasin-containing
medium (G).
The most striking difference appears in the first 36 h of development. During
this period, [3H]leucine incorporation rises steadily in the controls (M), to a
value of 200 dpm/gemmule, while in gemmulostasin-containing medium there is
a high peak of incorporation rising to about 500 dpm/gemmule. From the 42nd
to the 60th hour, the curves are more or less parallel; later, incorporation in
the conditioned medium falls well below its value in the mineral medium.
Biochemical control of sponge development
291
Figs. 2-3. Nuclear incorporation of [3H]thymidine into sponge gemmules after 36 h
of incubation at 20 °C. (A) Focusing on the nuclei; (B) focusing on the traces.
We may conclude from this result that the overall effect of gemmulostasin i&
not an inhibition but, on the contrary, a striking enhancement of the incorporation of [3H]leucine into proteins.
Effects of gemmulostasin upon the incorporation of labelled uridine into RNA
As seen in Fig. 5, the results obtained with [3H]uridine are essentially parallel
to those obtained with labelled leucine.
In the mineral medium (M), incorporation rises slowly during the first 36 h of
development, with a very steep increase later, while in the conditioned medium
(G) we find a high peak of incorporation at the 24th hour and, from the 48th
hour onwards, an incorporation rate that is well below that of the controls.
292
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Fig. 4. Radioactivity of protein extracts from gemmules during their incubation in
mineral medium ( # , M) and in conditioned medium ( O , G), after a 12 h pulse.
DISCUSSION
It should be recalled that from the point of view of the chronology of gemmulostasin action, the development of a gemmule can be divided into two phases.
During the first half of development, while they are sensitive to gemmulostasin, no morphological change can be detected in the cells. During the second
half of development, however, mitoses begin and the gemmule becomes insensitive to gemmulostasin (Rozenfeld, 1970). For the gemmules we are concerned
with here, incubated at 20 °C, the transition between the two phases lies somewhere around the 42nd hour of incubation.
Biochemical events in the uninhibited gemmule
These experiments show that in the absence of gemmulostasin, the gemmules
begin incorporating [3H]thymidine into their nuclear DNA at the end of the
first phase of development, i.e. before the mitoses begin. During the second
phase, the incorporation rate stays at a constant high level. It is reasonable to
suppose that the onset of [3H]thymidine incorporation corresponds to the pre-
Biochemical control of sponge development
293
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Time of incubation at 20 °C (h)
Fig. 5. Radioactivity of RNA extracts from gemmules during their incubation in
mineral medium ( # , M) and in conditioned medium (O, G), after a 12 h pulse.
prophasic synthesis of DNA, preparatory to the first wave of mitoses, and
that the plateau during the second phase is accounted for by the asynchrony of
the mitoses during this phase (Rozenfeld, 1970).
The incorporations of [3H]uridine and [3H]leucine rise slowly and steadily at
the end of thefirstphase, reach a high peak at the beginning of the second phase
and remain high. The cell divisions themselves, the digestion of the vitelline
platelets in many cells and finally, the digestion of the micropyle membrane
(Rozenfeld, 1971; De Vos & Rozenfeld, 1974) probably imply the synthesis of
new proteins and, accordingly, the synthesis of RNA involved in this process.
294
F. ROZENFELD
Biochemical events in the presence of gemmulostasin
The most salient effect of gemmulostasin is to erase the peak of [3H]thymidine
incorporation that normally occurs at the end of the first phase of development,
and to keep it low from then on. In so far as the peak corresponds to the preprophasic synthesis of DNA, this means that it is at this level that gemmulostasin inhibits the first wave of mitoses of normal development. Such an effect
would be consistent with the fact that the overall role of this agent is to block
development, and that a wave of karyokineses is the first morphological event of
this development (Brien, 1932; Wierzejski, 1935; Berthold, 1969; Rozenfeld,
1970).
During the second phase of development, incorporation of [3H]uridine and of
3
[ H]leucine is depressed, though not completely inhibited. This result is to be
expected if part of the protein and RNA syntheses is related to the mitoses
implied in the morphological changes of the cells originating from these mitoses.
The residual incorporation during that phase is clearly related to biochemical
events that do take place even in the absence of any morphological change. It has
indeed been observed in our laboratory (Rasmont, personal communication)
that the gemmules of Ephydatia fluviatilis, even when they are kept from germinating by the action of cold, show signs of ' ageing' that should be accounted for
in terms of biochemical events continuing under such conditions.
The most interesting of our results, however, relate to the first phase of
development, during which a high peak of incoroporation of [3H]uridine and
[3H]leucine occurs.
This overall enhancement of the incorporation into RNA and protein fractions is, to our present knowledge, the only positive reaction of the gemmules to
gemmulostasin. It should be stressed that it occurs during the gemmulostasinsensitive phase of development, and parallels an inhibition of tritiated thymidine
into DNA. It is therefore highly probable that some causal relation exists
between these various phenomena. Several hypotheses might account for such
a relationship. Before we choose between these hypotheses, we should know how
the syntheses of DNA, RNA and protein are directly affected by gemmulostasin. Indeed, in the present state of the investigation, it is not excluded that the
modifications brought about in incorporation into these macromolecules could
be a result of modifications in the specific activities of their precursors, e.g.
through an alteration of the properties of cell membranes by gemmulostasin.
Our laboratory is presently carrying on the purification and chemical characterization of gemmulostasin. The result ofthat work may eventually enable us to
resume and carry further the present research, with known concentrations of
pure and, possibly, labelled inhibitor.
Biochemical control of sponge development
295
I wish to thank Professor R. Rasmont for suggesting the problem, for advice and encouragement during the research and for criticism in the preparation of the manuscript. I am grateful
to Drs C. De Vos and G. Urbain, who helped with liquid scintillation counting at the Laboratoire de Physiologie animale.
The assistance of Mrs N. Van Mol with the preparation of diagrams and Mr A. Weisman
with the photographs is gratefully acknowledged.
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{Received 20 September 1973, revised 29 January 1974)