J. Embryol. exp. Morph. Vol. 17, 3, pp. 473-80, June 1967
With 2 plates
Printed in Great Britain
473
Promoting activity of extracellular materials
on sponge cell reaggregation
By A. P. MACLENNAN 1 & R. Y. DODD 1
Microbiological Research Establishment, Porton
INTRODUCTION
It has long been known that sponge cells exhibit specific reaggregation
(Wilson, 1907), manifested by the production from mixed cell suspensions of
separate aggregates containing cells originating from one species only. A
possible mechanism for this process is the specific adhesion of cell surfaces.
Humphreys (1963) and Moscona (1963) showed that prolonged washing of
sponge tissues with a Ca2+- and Mg2+-free sea-water medium yielded cells
unable to reaggregate at low temperature. However, the supernatant fluid from
this washing procedure contained a labile component that restored the capacity
of the cells to reaggregate in the presence of divalent cations at low temperatures.
Moreover, this activity was species specific in the system Microciona prolifera/
Haliclona oculata inasmuch as the supernatant fluid from Microciona cells would
cause Microciona aggregates alone to be formed from a MicrocionajHaliclona
cell mixture, whereas Haliclona supernatant fluid would selectively promote
Haliclona aggregation.
It has been reported by MacLennan (1963) that the antigenic character of the
sponge cell surface is dominated by specificities which are shared with heteropolysaccharide substances that can be extracted directly from sponges. Since the
selective promotion of reaggregation (Humphreys, 1963; Moscona, 1963)
implies a recognition, by the extracellular product, of a complementary cell
surface component in homologous cells, it can be argued that the specific
carbohydrate antigens of the cell surface function as such complementary
components. As a first stage in the study of this postulated interaction of extracellular reaggregation-stimulating factor and cell surface, we have examined
sponges of known serological surface character for their ability to produce
substances with reaggregation stimulating activity.
1
Authors' address: Microbiological Research Establishment, Porton, near Salisbury,
Wilts, U.K.
30
J E E M 17
474
A. P. MACLENNAN&R. Y. DODD
MATERIALS AND METHODS
The siliceous sponges Halichondria panicea, Hymeniacidon perleve, Cliona
celata, Axinella sp., Ophlitaspongia seriata, Pachymatisma johnstonii, Stylostichon plumosum, Ficulina (Suberites) ficus and the calcareous sponge Grantia
compressa were collected from South Devon coastal waters. They will be referred
to by their generic names alone. The sponges were collected into sea water at
0-4 °C or placed in plastic bags, gassed with oxygen and stored at 0-4 °C. All
sponges were used within 24 h of collection.
The procedures described by Humphreys (1963) were used to prepare
'chemically dissociated cells' and 'Aggregation Factor' solutions. The cells
were obtained by washing the sponge tissue, cut into 3 mm3 pieces, in 20 vol.
of cold Ca2+- and Mg2+-free artificial sea water (CMF) for 30 min at 0-4 °C.
The pieces of sponge were then squeezed through nylon net (100 /JL mesh) into a
further 20 vol. of cold CMF. The resultant suspension was centrifuged at
700 g for 10 min and the sedimented cells resuspended in 10 vol. of cold CMF.
This suspension was agitated gently for 6 h at 0-4 °C, after which the cells were
again sedimented and finally suspended in a complete sea-water medium (SW)
at a concentration of 20 x 106 cells/ml, as determined by counting in a FuchsRosenthal or Thoma chamber. At this stage the degree of dissociation of the
cells could be estimated. Small aggregates of five or six cells occurred with a
frequency of about 1 per 300 single cells. Smaller or larger groups of cells were
less frequently observed. The microscopic appearance of the cell suspensions was
similar to that illustrated in Plate 1, figs A and C. Viability was not determined.
Aggregation Factors were prepared by washing the sponge tissue, cut into
1 cm3 pieces in four changes of 7-5 vol. of cold CMF, each of 10 min. The
pieces were then squeezed through nylon net into 2-5 vol. of cold CMF and
stirred at 0-4 °C for 4 h. The suspension was lightly centrifuged to remove cells,
then at 30000 g for 1 h to yield a clear, faintly coloured supernatant fluid that
constituted the Aggregation Factor preparation. Ca 2+ was added to 2 mM to
stabilize activity. In parallel with the preparation of Aggregation Factors,
Control Factors were produced using SW in place of CMF throughout. For each
experiment fresh Aggregation Factor was prepared at the same time as the
chemically dissociated cell suspensions and was used within 2h. Over this
period it was kept at 0-4 °C.
Tests for reaggregation were carried out using the rotation-mediated system
introduced by Moscona (1961). Volumes (1-5 ml) of chemically dissociated cell
suspension were placed in 25 ml stoppered conical flasks, together with an
equal volume of Aggregation Factor or Control Factor. Serial twofold dilutions
of the Factors in SW were also tested to determine the end-point of activity,
which was expressed in terms of the final dilution of Aggregation Factor in
the cells plus Factor mixture. As controls on the efficiency of the dissociation
procedure, mixtures of chemically dissociated cells and SW alone were tested;
Promotion of sponge cell aggregation
Al5
these preparations represented the base-line against which promotion of aggregation was estimated. Flasks were mounted on a gyratory shaker (New Brunswick
Scientific Co., New Brunswick, N J . ; modified model S-3S with variable speed
control) and shaken at 80 rev/min, at 0-4 °C.
Aggregation was estimated after 16 h on the shaker. At this time aggregation
had been at its maximum value for several hours. Deterioration of the aggregates,
associated with the appearance of bacteria in the preparations, did not normally
begin until after 20 h.
The degree of aggregation was estimated macroscopically. This visual
scoring on a 0 to + + + + system was associated with particular microscopic
appearances in the following manner:
0
+
+ ++ +
A turbid suspension of free cells (see Plate 1, fig. A).
A granular suspension of small aggregates (0-025-0-1 mm diameter).
1-3 large aggregates 0-5-2-0 mm. diameter) (see Plate 1, fig. B).
The transition from + to + + + + represents a gross visual difference within
which intermediate stages could fairly easily be defined, both visually and by
microscopic counting and measurement. However, finer distinctions than those
represented by 0, + and + + + + are not required to sustain the conclusions
drawn in the present paper.
The term 'feeble activity' in reference to Aggregation Factor signifies that
the undiluted solution gave less than a + + + + reaction.
RESULTS
Activity of Aggregation Factors
In all but three of 35 batches of Hymeniacidon collected over a period of one
year, Aggregation Factor preparations promoted the reaggregation of chemically
dissociated homologous cells. Activity was found at dilutions as high as 1/32,
comparing favourably with Microciona activities (Humphreys, 1963). Activity
was said to be present when aggregation was distinctly greater than that in the
SW controls which frequently showed a + reaction. Although 3/35 tests were
invalidated by the concurrence of feeble Aggregation Factor activity with control
reactions greater than + , in the remaining 32 tests + + + + reactions were
given by Aggregation Factors throughout the twofold to eightfold range of
dilutions, against Control Factor reactions of + or less (see Plate 1).
To determine whether or not activity differences between Hymeniacidon
Aggregation Factor and Control Factor preparations were based on gross
chemical differences some simple analyses were performed. Table 1 records the
results for three such pairs of preparations.
Because of the low and similar values for organic phosphorus contents, and for
260 mjti absorptions, of Aggregation and Control Factors, it appeared unlikely
that aggregation-promoting activity was due to nucleic acid or nucleoprotein
30-2
476
A. P. MACLENNAN & R. Y. DODD
acting in the manner suggested by Steinberg & Roth (1964). Pretreatment of
Hymeniacidon Aggregation Factor at 37 °C for 30 min with ribonuclease
(Boerhinger and Soehne, 42 K units/mg.) or deoxyribonuclease (Seravac Laboratories), each at 20 /^g/ml reaction mixture, did not decrease Aggregation Factor
activity. Moreover, highly polymerized preparations of calf thymus DNA
(Seravac Laboratories) and bacterial ribosomal RNA, dissolved in SW to give
organic phosphorus concentrations corresponding to that of Hymeniacidon
Aggregation Factor, did not promote aggregation.
Table 1. Composition of Hymeniacidon Aggregation Factor
and Control Factor (in mg/ml)
Preparation
(1) Aggregation Factor
Control Factor
(2) Aggregation Factor
Control Factor
(3) Aggregation Factor
Control Factor
Activity*
16
32
16
Dry
weight f
0-78
0-56
0-44
0-42
0-80
0-38
Protein X
104
100
0-58
0-78
0-86
0-64
Carbohydrate§
008
007
007
006
007
004
Bound
0005
0007
0004
0005
0005
0003
* Reciprocal of highest dilution showing activity.
t Sample dialysed thoroughly and freeze dried.
t Lowry method, Bovine Plasma Albumin standard (Lowry et al. 1951).
§ Orcinol method, glucose standard (Synge & Wood, 1958).
|| See King (1951).
The overall similarity in composition of Aggregation Factor and Control
Factor was supported by diffusion-precipitin analysis in agar using antisera
to Hymeniacidon cells or to polysaccharide antigens (MacLennan, 1963). Five
antigens were detected, common to both preparations and present in roughly
equal amounts as judged by the lines of precipitation formed by serially diluted
preparations (Plate 2). The antigens present included the thermostable polysaccharides with cell surface specificities (MacLennan, 1963).
Of the nine species of sponge examined, Hymeniacidon consistently showed
high Aggregation Factor activity and Halichondria was inactive in six tests,
feebly active in six others. Of the other sponges, in exploratory tests, all but
Grantia and Pachymatisma showed Aggregation Factor activity for homologous
cells. However, activity was feeble, or variable from batch to batch, and its
assessment was complicated by the considerable spontaneous reaggregation of
chemically dissociated cells in the absence of added Aggregation Factor.
Specificity of Aggregation Factors
Single species reaggregation
In contrast to the 'species specificity' of Aggregation Factor activity previously reported for other sponges (Humphreys, 1963; Moscona, 1963) Hymenia-
/. Embryo!, exp. Morph., Vol. 17, Part 3
PLATE 1
1 mm
1 mm
PLATE 1. Rotation-mediated aggregation of chemically dissociated sponge cells in SW at
4°C.
Fig. A. Hymeniacidon cells plus Hymeniacidon Control Factor.
Fig. B. Hymeniacidon cells plus Hymeniacidon Aggregation Factor—single large aggregate.
Fig. C. Halichondria cells plus Hymeniacidon Control Factor.
Fig. D. Halichondria cells plus Hymeniacidon Aggregation Factor—single large aggregate.
A. P. MACLENNAN & R. Y. DODD
facing p. 476
J. Embryol. exp. Morph., Vol. 17, Part 3
A. P. MACLENNAN & R. Y. DODD
PLATE 2
Promotion of sponge cell aggregation
Ml
cidon Aggregation Factor promoted the aggregation of chemically dissociated
cells of Halichondria (Plate 1) and Ficulina, giving titres similar to those observed
with Hymeniacidon cells. Control Factor was inactive against the cells of all
three species.
In reciprocal tests Halichondria Aggregation Factor feebly promoted the
aggregation of Hymeniacidon cells in 3/11 tests; in these positive tests feeble
activity against Halichondria cells was also shown. Halichondria Control Factor
was always inactive.
Mixed species reaggregation
To determine' normal' reaggregative potential and specificity, cell suspensions
of Hymeniacidon and Halichondria were prepared precisely as described under
Materials and Methods, but with SW in the place of CMF. In the rotationmediated system at low temperature a mixture of the two cell suspensions sorted
out into orange {Hymeniacidon) and yellow-green {Halichondria) aggregates.
Hymeniacidon and Ficulina mixtures behaved similarly. In both mixtures
aggregation did not reach the + + + + stage, and the species-specific character
of the aggregates was best observed microscopically.
Segregation into small species-specific aggregates ( + stage) was occasionally
detectable even in mixture of cells that had been chemically dissociated and then
returned to SW. The influence of Hymeniacidon Aggregation Factor on this
aggregation was examined. The mixed cell suspension plus Hymeniacidon
Aggregation Factor reached an intermediate stage of aggregation into species
specific aggregates at 1-2 h. By 16 h a single aggregate or a small number of
large aggregates had formed, at dilutions of Aggregation Factor that produced
this + + + + effect in the cell suspensions of each species tested separately. The
single aggregates were not species-specific, since they would have been readily
distinguishable by colour alone.
In other experiments separate cell suspensions of Hymeniacidon, Halichondria
and Ficulina were allowed to aggregate to beyond the + stage in the presence of
Hymeniacidon Aggregation Factor. When the Hymeniacidon aggregate suspension was then mixed with each of the other two, aggregation proceeded by the
PLATE 2. Diffusion-precipitin analysis of Hymeniacidon Aggregation Factor and Control
Factor.
Fig. A. Reservoirs 1, 3 and 5 contain 10, 2 and 0-4mg/ml respectively of a Hymeniacidon
Aggregation Factor. Reservoirs 2, 4 and 6 contain 10, 2 and 0-4mg/ml respectively of
Hymeniacidon Control Factor. The central reservoir contains a rabbit antiserum against
Hymeniacidon cells.
Fig. B. All antigen preparations were heated, as lOmg/ml aq. solns, for 2 h at 100 °C.
Reservoirs 1, 3, 5 and 7 contain 10, 2, 0-4 and 0-1 mg/ml of a Hymeniacidon polysaccharide
preparation (MacLennan, 1963). Reservoirs 2 and 6 contain Hymeniacidon Aggregation
Factor, reservoirs 4 and 8 contain Hymeniacidon Control Factor, all at 10 mg/ml. The two
central reservoirs contain a rabbit antiserum against Hymeniacidon polysaccharide preparation.
478
A. P. MACLENNAN & R. Y. DODD
incorporation of these specific aggregates into a final single mixed aggregate of
intermediate colour.
Non-specific agglutination of sponge cells
The importance of including Control Factors in the study of Aggregation
Factors was illustrated by the behaviour of Axinella. The Aggregation Factor of
this sponge had an apparent broad reactivity with cells of other species in
addition to its own. Control Factor, however, showed the same cross-reactions,
but was inactive against Axinella cells. Thus, heterologous activity, extractable
in the presence of divalent cations, can also occur together with Aggregation
Factor and complicate the interpretation of experiments. The cross-reactivity
extends to an agglutination of fowl and mammalian erythrocytes and is probably
related to the heteroagglutination of sponge cells by foreign sponge extracts
described by Galtsoff (1929).
DISCUSSION
The demonstration of an aggregation-promoting factor in Hymeniacidon,
extractable only in the absence of divalent cations, supports the earlier discovery
of such factors in Microciona prolifera and Haliclona oculata (Humphreys,
1963; Moscona, 1963). Chemical and serological analysis of Aggregation Factor
and Control Factor preparations suggest that the active principle is a minor
component of the total extract.
The difference in specificity of Aggregation Factor activity in the Microciona/
Haliclona system on the one hand, and the Hymeniacidon/Halichondria/Ficulina
system on the other, may prove to be an expression of the closeness of taxonomic
relationship within the systems; although the taxonomy of the Demospongiae
is by no means clear, it is possible to state that Hymeniacidon and Halichondria
or Hymeniacidon and Ficulina represent families within an order, whereas
Microciona and Haliclona represent two distinct orders (Levi, 1957; Hartman,
1958). Moreover, in support of conventional criteria of relationship, Hymeniacidon, Halichondria and Ficulina cross-react strongly with respect to agglutination by antisera (A. P. MacLennan, unpublished observation), implying a
similarity in the structure of the cell surfaces that may prove relevant to the
observed common promotion of aggregation by Hymeniacidon Aggregation
Factor (see Yazikov, 1965).
The observed segregation of Hymeniacidon, Halichondria and Ficulina cells
from unpromoted mixtures, while each alone is promoted by Hymeniacidon
Aggregation Factor, raises the interesting possibility that sponge cell reaggregation, at least in the species tested, exhibits a specificity at two levels:
first, a broad specificity demonstrated by an extracellular reaggregationstimulating material acting in this case on three closely related sponges; secondly,
a narrow, or species specificity, presumably vested in a cell surface component
and responsible for the segregation of cells originating from different species.
Promotion of sponge cell aggregation
479
This narrower selectivity may be suppressed or overcome in the presence of
heterologous Aggregation Factor of high activity.
It may appear that Hymeniacidon Aggregation Factor is entirely non-specific
in activity, sharing only with the previously described specific Aggregation
Factors an attachment to the cell that is dependent on the presence of divalent
cations. However, preliminary observations (MacLennan & Dodd, unpublished
observations) suggest that promoting activity may not extend to antigenically
unrelated species of sponge.
SUMMARY
1. A number of species of sponge showed enhanced aggregation behaviour
in the presence of cell extracts prepared under denned conditions previously
found suitable for other sponges.
2. The promoting activity of one such preparation appeared to be based on a
minor component of the total extract.
3. The specificity of promotion was of a low order and led to mixed aggregate
formation from species that segregated specifically in unpromoted mixtures.
4. The observations are discussed in relation to the known antigenic relationships of the cell surfaces of the species examined.
RESUME
Activite favorisante de substances extacellulaires sur la reagregation
de cellules d'eponges.
1. Plusieurs especes d'eponges ont presente une meilleure ragregation en
presence d'extraits cellulaires prepares dans des conditions definies, prealablement connues comme favorables pour d'autres eponges.
2. L'activite favorisante d'une de ces preparations s'est revelee due a un
composant mineur de l'extrait total.
3. La specificite de cette action etait faible et a conduit a la formation
d'agregats mixtes a partir d'especes qui se segregaient specifiquement dans
des melanges non favorises ou peu favorises.
4. Ces observations sont discutees dans leur relations avec les rapports
antigeniques connus entre surfaces cellulaires des especes examinees.
We thank Mr H. E. Wade for a gift of bacterial ribonucleic acid, and Miss J. Legg for
valuable technical assistance.
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{Manuscript received 11 July 1966, revised 14 January 1967)