249
IODINE COMPOUNDS AND FERTILISATION
V. AGGLUTINATION AND CHEMOTAXIS OF THE SPERM
B Y G. S. CARTER,
Corpus Christi College, Cambridge.
(From the Laboratory of Experimental Zoology, Cambridge, and the Marine
Laboratory, Millport.)
(Received if>th December, 1931.)
IF fresh and ripe spermatozoa of E. esculentus or E. miliaris are put into a solution
of thyroxine in sea-water of a concentration of 1/50,000 by weight and at the hydrogen-ion concentration of sea-water (/>H 8-2), they very soon come together by their
heads and form clumps. The most favourable concentration of the sperm for
observing this phenomenon is a mixture of sea-water and undiluted sperm in a
proportion of 500 : 1 or 1000 : 1. This "agglutination" is visible under the microscope in 1-3 min. and becomes very obvious in 10 min. In a short time the larger
clumps fall to the bottom and there form a flocculent precipitate, which can be
collected into the centre of the dish by shaking, and is easily visible to the naked
eye. The clumps are dense and at first spherical, but their shape becomes irregular
as they grow larger, probably in part by the coalescence of several small clumps to
form one larger one, and in part by uneven addition of spermatozoa on the outside
of the clump. The sperm are active, at least for many minutes, after they are
agglutinated. The agglutination is permanent. It only occurs in fresh and ripe
sperm. Perfectly ripe sperm may show a slight spontaneous agglutination in seawater, but this is always much less than the agglutination in the presence of thyroxine.
It can be easily seen that most of the clumps of spermatozoa have at their
centres crystals of the precipitate which forms in a 1/50,000 solution of thyroxine
(1931 a, p. 179). This is almost always true of the larger clumps. It is, however,
certainly not true that the agglutination can only occur round the crystals. In the
early stages of the agglutination the sperm often come together by twos or threes,
and frequently without any connection with the crystals. Further, if the crystals
are removed by centrifuging the solution or by filtering it through a sintered glass
filter, agglutination still occurs, although it is far less obvious and the clumps' are
much smaller and looser1. Clearly the agglutination is caused in part by some
substance in true or colloidal solution.
Owing to the formation of the precipitate in solutions of thyroxine in sea-water,
it is not possible to give an accurate figure for the minimum concentration of
1
It is less in a solution of thyroxine filtered through a filter paper, perhaps because the dissolved
thyroxine is adsorbed on the paper.
JEB-Ixiii
16
250
G. S. CARTER
thyroxine in which agglutination occurs. It was observed in a solution made by
adding 1/50,000 of thyroxine to sea-water, both in the presence of the crystals
and after they had been removed, and in both these solutions diluted with twice
their volume of sea-water. It was never certainly observed when the dilution was
greater than this. These solutions are of the same order of concentration as those
found to be necessary to produce the effects of thyroxine on the activity of the
sperm. They are much more concentrated than those needed to improve the
development of washed eggs. The cause of this difference has been discussed in
a previous paper (1932 a, p. 247). In the present experiments it was necessary
to use a fairly dense suspension in order that the sperm might agglutinate.
Neither des-iodo-thyroxine nor any of the series of related chemical substances
which were used for comparison with thyroxine (1931 a, p. 179) produced this
agglutination. Adrenaline was also ineffective.
The agglutination is almost equally marked in the sperm of the two species of
Echinus, and is of the same character in both.
Lillie (1914) observed that agglutination of the sperm of Nereis and Arbacia
occurs in alkaline sea-water. This is also true of the sperm of Echinus, but the
hydrogen-ion concentration at which it occurs varies greatly with the ripeness
of the sperm. It has been already mentioned that in very ripe sperm it may occur
to a slight extent in sea-water of normal alkalinity, but in sperm which is not ripe
far more alkalinity is required. For instance, in one experiment, sperm of Echinus
esculentus gave a very slight agglutination in sea-water + 1 per cent. Njio NaOH,
and a strong agglutination in sea-water + 2 per cent. iV/10 NaOH (pH of both
these media more alkaline than 9-4), but it agglutinated strongly in sea-water + thyroxine (1/50,000) at pH 8-2. It is clear that only experiments in which the pH of
the medium is accurately controlled are of value in estimating the effect of thyroxine
in causing agglutination. This was always done in the experiments here discussed.
The agglutination in alkaline sea-water is loose and the clumps irregular. It
is far looser than in the presence of thyroxine, especially when the crystals are
present. Also, in alkaline sea-water, the sperm are rendered immobile almost
immediately by the alkalinity.
In spite of these differences thyroxine resembles alkalinity in causing agglutination. Here, as in the phenomena discussed in a previous paper (1931 a, pp. 185,
191), there is a parallel between the effects of the presence of thyroxine and of
changes in the hydrogen-ion concentration of the medium.
In being permanent, the agglutination produced by thyroxine resembles
Lillie's hetero-agglutination rather than his iso-agglutination (1913). It resembles
hetero-agglutination also in that the sperm remain active for some time after they
are agglutinated1, in the spherical form of the clumps and in the tightness of the
agglutination in the clumps. All these characters are more marked when the
crystals are present in the medium.
1
Sampson (1922) observed this in the sperm of Kathanna. Hetero-agglutination is usually
toxic (Lillie, 1913). The agglutination caused by thyroxine is also toxic after the sperm has been
agglutinated for some minutes.
Iodine compounds and fertilisation
251
The fact that many of the clumps are formed round these crystals shows that
there is some chemotaxis of the sperm towards the crystals. The sperm does not
form clumps round other bodies such as free cells from the testis and the various
granules which are often present in the suspension at the same time. These may be
seen without any covering of sperm after the agglutination has occurred. The
formation of the clumps cannot, therefore, be caused simply by the head of the
sperm, having become adhesive by the action of thyroxine, coming into contact
with the surface of the crystal. Some chemotaxis must be present, and, as the
substance forming the crystals is almost certainly closely related to thyroxine
(1931 a, p. 179), this chemotaxis may be added in all probability to the other effects
of the drug.
However, chemotaxis is poorly developed in the sperm of Echinus. Attempts
to repeat Lillie's experiments (1914), in which a drop of egg-water was introduced
into a suspension of the sperm under a coverslip, failed to give definite results.
Neither egg-water nor a solution of thyroxine caused the formation of a ring of
spermatozoa surrounding the drop.
Agglutination of the same type as that described in the sperm of Echinus was
observed in the sperm of Solaster papposus in a solution of thyroxine in sea-water
at a concentration of 1/50,000 and in this solution diluted with its own volume of
sea-water. It was also observed in the sperm of Solaster endeca and Asterias rubens
at a concentration of 1/50,000 and, perhaps, slightly at lower concentrations. The
sperm of Buccinum undatum was found to agglutinate very strongly. Fertilisation
is internal in this animal, and sperm taken from the genital duct is rarely active on
dilution in sea-water. In experiments in which it did activate the sperm was found
to agglutinate in a solution of thyroxine at a concentration of 1/50,000 and in this
solution diluted with ten times its volume of sea-water.
Of the series of chemical substances related to thyroxine mentioned above
(p. 250) only tyrosine, tryptophane, iodine and potassium iodide were used in
experiments on the sperm of these animals. They did not cause agglutination.
In spite of the small number of these observations, they serve to indicate that
the action of thyroxine in producing agglutination is not confined to the Echinoidea.
It is apparently a wide-spread phenomenon among the marine invertebrates, and
occurs in sperm which normally fertilises the egg within the body of the parent.
It remains to consider how far these results confirm the conclusion of previous
papers (193 ib, 1932 a) that thyroxine is chemically related to one of the components
of the egg secretions.
It is well known that permanent agglutination of sperm may be caused by many
substances besides the constituents of egg-water. For example, Gray (1915) found
that trivalent ions could cause agglutination. It has been mentioned above that
alkalinity of the medium may do so.
The type of agglutination described in this paper differs in certain respects
from that due to other chemical substances, especially in the closeness of the
agglutination and in the activity of the agglutinated sperm. It is peculiar in that it
is strongest around the crystals which form in the solution of thyroxine, but it
16-a
252
G. S. CARTER
has been shown above that this is probably due to chemotaxis towards these
crystals. There are no similar crystals in the other chemical solutions which cause
agglutination.
In the earlier stages of the agglutination, and particularly after the crystals have
been removed, there is very little difference between this type of agglutination and the
hetero-agglutination caused by egg-water (cf. Sampson, 1922). Nevertheless, the facts
of agglutination do not give conclusive evidence that thyroxine is more closely related
to a constituent of the secretions than the other substances which cause agglutination.
That conclusion was suggested by the very different phenomena discussed in
the earlier papers of this series, and its truth must rest on evidence derived from
these phenomena and other evidence to be given in later papers of this series. It
can, however, be said that the facts of agglutination are not in conflict with such a
theory, and that these facts make it probable that, if thyroxine is related to a component of the secretions, it is to one of the agglutinins that it is related.
All the types of agglutination caused by chemical substances, and among them
that caused by thyroxine, resemble hetero-agglutination, rather than iso-agglutination, in being permanent. So far as thyroxine is concerned, this is to be expected,
for thyroxine is known to cause this type of agglutination in sperm of several
distantly related animals and the iso-agglutinin, in contrast to the hetero-agglutinin, is specific in its action. If there is any chemical relatedness between a constituent of the secretions and thyroxine, these experiments indicate a closer relationship with the hetero-agglutinin than with the iso-agglutinin. This again is to be
expected, for the iso-agglutinin is probably a very complex body and far more
complex than thyroxine (Lillie, 1914, Richards and Woodward, 1915).
Whether these conclusions can be brought into line with those derived from
the results of the experiments on the egg, will be discussed in a later paper.
SUMMARY.
1. Agglutination occurs in solutions of thyroxine in sea-water. This agglutination is permanent, and occurs especially, but not only, around the crystals
which are present in solutions of thyroxine. It was observed in the sperm of species
of Echinus, Solaster, Asterias and Buccxnum.
2. There is chemotaxis of the sperm towards these crystals.
3. The resemblance of this type of agglutination to hetero-agglutination is
discussed.
REFERENCES.
CARTER, G. S. (1931 a). Journ. Exp. Biol. 8, 176.
(1931 *)• Journ. Exp. Biol. 8, 194.
(1932 a). Journ. Exp. Biol. 9, 238.
GRAY, J. (1915). Quart. Journ. Mxcr. Sci. 61, 119.
LILLIE, F. R. (1913). Journ. Exp. Zool. 14, 515.
(1914). Journ. Exp. Zool. 14, 523.
RICHARDS, A. and WOODWARD, A. E. (1915). Biol. Bull. 28, 140.
SAMPSON, M. M. (1922). Biol. Bull. 43, 267.