1. Bracken spermatozoids are chemotactically attracted by malic acid salts and in a pH gradient aggregate between pH 3.5 and 5.6.



2. This response to pH occurs only when the sperm suspension contains malate.



3. At higher pH's, the response to an H+ gradient is as strong as the response to a malate gradient.



4. These observations suggest that bracken spermatozoids are sensitive to the bimalate ion rather than the malate ion. This is consistent with an earlier suggestion that chemotactic activity is associated with the cis-configuration, for the bimalate ion will have this configuration, while the malate ion and the malic acid molecule will have the trans-configuration

Brokaw, C. J.

English

Fern spermatozoids respond chemotactically to concentration gradients of malic or maleic acid salts (Pfeffer, 1884). They respond weakly to salts of a few other dicar-boxylic acids, but do not respond to succinate or fumarate (Shibata, 1911; Rothschild, 1956). Previous workers concluded, without adequate justification, that the malate ion, and not the undissociated malic acid molecule, is chemotactically active; but no consideration has been given to the possibility that the bimalate ion may be the active species. A malate-dependent aggregation of bracken spermatozoids in a pH gradient has been briefly reported (Brokaw, 1957). In this paper, this phenomenon and several related observations are examined in greater detail. MATERIAL AND METHODS Spores of bracken, Pteridium aquilinum (L.) Kuhn, were sown thickly on agar plates made up with approximately 1 % agar in a Knop&apos;s solution (o-i % KNO3, 0-05

C. J. Brokaw

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Chemotaxis of bracken spermatozoids: The role of bimalate ions

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CHEMOTAXIS OF BRACKEN SPERMATOZOIDS
THE ROLE OF BIMALATE IONS
BY C. J. BROKAW
Department of Zoology, University of Cambridge
(Received 10 August 1957)
Fern spermatozoids respond chemotactically to concentration gradients of malic or
maleic acid salts (Pfeffer, 1884). They respond weakly to salts of a few other dicar-
boxylic acids, but do not respond to succinate or fumarate (Shibata, 1911;
Rothschild, 1956). Previous workers concluded, without adequate justification, that
the malate ion, and not the undissociated malic acid molecule, is chemotactically
active; but no consideration has been given to the possibility that the bimalate ion
may be the active species.
A malate-dependent aggregation of bracken spermatozoids in a pH gradient has
been briefly reported (Brokaw, 1957). In this paper, this phenomenon and several
related observations are examined in greater detail.
MATERIAL AND METHODS
Spores of bracken, Pteridium aquilinum (L.) Kuhn, were sown thickly on agar plates
made up with approximately 1% agar in a Knop's solution (o-i% KNO3, 0-05%
NaCl, 0-05% CaSO4, 0-05% MgSO4, 0-05% Ca3(PO4)2). A dense mat of small
prothalli was obtained after about 3 months' growth. When a piece of this mat is
removed, placed in a small quantity of water, and gently squashed with a glass rod
to ensure thorough wetting, spermatozoids emerge from the antheridia. After about
20 min. the suspension of spermatozoids may be removed with a pipette.
A cleaner and/or more concentrated sperm suspension can be obtained by taking
advantage of the chemotactic reactivity of the spermatozoids. A drop of sperm
suspension is placed on a glass plate under a low-power microscope and small
droplets of sodium L-malate solution are carefully injected into the drop of sperm
suspension. After 3-5 min., the aggregations of spermatozoids in these droplets
may be removed with a fine-tipped pipette. This procedure may be repeated to
obtain specially clean sperm suspensions for dark-ground photomicrography.
The crude sperm suspension obtained from the prothalli is contaminated with
substances which act as pH buffers and with traces of chemotactically active sub-
stances. When a low activity of malate is required in the sperm suspension, or when
accurate pH control is necessary with minimum concentrations of buffer, the sperma-
tozoids must be washed several times by centrifugation and resuspension in buffer.
This procedure causes some damage to the spermatozoids, which must not be
confused with the effects of low malate concentration.
Chemotaxis of bracken spermatozoids
DISSOCIATION OF MALIC ACID
The dissociation of malic acid can be represented by
The relative concentrations of malic acid, HjM, bimalate ion, HM~, and malate ion,
M2-, existing at any pH can be calculated and are shown by the curves in Fig. i.
4 5 6 7 8
PH
Fig. i. Relative concentrations of malate species (logarithmic scale) at various pH's
There are two forms of bimalate ion, depending on which carboxyl group is
ionized. Consideration of the pK's of a- and /3-hydroxy-butyric acids (39 and 4-5)
indicates that bimalate will consist of about; 80% of the form shown in Fig. 2 a and
about 20 % of the form shown in Fig. 2b. In either form, the possibility of hydrogen
bonding between the dissociated and undissociated carboxyl groups will tend to
favour the m-configuration. A hybrid structure (Fig. 2 c) may best represent the
bimalate ion, although there is no evidence that it is appreciably stabilized. On the
other hand, where the two carboxyl groups are both neutral, as in the malic acid
molecule (Fig. 2 d), or both negatively charged, as in the malate ion (Fig. 2e), the
fronj-configuration will be strongly favoured, so that the two groups will be
separated from each other as much as possible.
RESULTS
A drop of o-1 M hydrochloric acid was placed on a glass slide in contact with a drop
of sperm suspension containing 0-005 M Zrw-(hydroxymethyl) aminomethane—
hydrochloric acid buffer, pH 8-i; 10-4 M sodium L-malate; and a small amount of
pH indicator. Under these conditions the acid diffuses into the sperm suspension,
neutralizes the buffer, and changes the colour of the pH indicator. A sharp colour
change and, therefore, a sharp pH change occur at the junction of the region in
which the buffer is completely neutralized with that in which it is not yet neutralized.
This junction moves slowly across the drop of sperm suspension as the acid diffuses
into the drop. A narrow aggregation of spermatozoids forms in this region of sharp
J3 Eip. Blol. 35, 1
194 C. J. BROKAW
pH change (Brokaw, 1957). Dark-ground track photographs of the spermatozoids
in this aggregation show them swimming actively within a narrow band about 75 /x
wide and turning sharply back into the band from both sides.
The same phenomenon was observed with sperm suspensions buffered with
o-oi M sodium citrate, sodium phosphate, or sodium borate buffers, at pH's ranging
from 5-6 to 87. With borate buffer, pH 87, and either cresol red indicator (pK 8-3)
or brom cresol purple indicator (pK 6-3), the band of spermatozoids was located in
HOCH
1
1
\
c
9
COO
COOH
HOOC'
.COOH
HOCH
H.A
^cooe
6
COOH
HOCH
1
^ C H ,
eooc
H
B
HOCH
CH,
/-COO
ocfr \
^coo
c
e
e
H
d t
Fig. 2. Configurations of malate species. For details see text.
the same position as the colour change of the indicator, which confirmed the existence
of a steep pH gradient in the region of sperm aggregation. With methyl-orange
indicator (pK 3-5), the colour change was located slightly on the acid side of the
band of spermatozoids. These experiments locate the region ' preferred' by bracken
spermatozoids between pH 3-5 and 5-6.
When the spermatozoids are repeatedly washed by centrifugation and re-
suspended in buffer solution without malate, no aggregation occurs when a pH
gradient is established as described above. If io"6 M sodium L-malate is added to
a repeatedly washed sperm suspension, the spermatozoids again aggregate in a band
when a pH gradient is established.
Fig. 1 shows that when a pH gradient extending from pH 2 to pH 8 is established
in a solution of uniform malate concentration, there is a maximum concentration of
the bimalate ion at pH 4-2 and bimalate gradients on either side of this point. The
chemotactic response of bracken spermatozoids to these bimalate gradients could
cause them to aggregate around pH 4-2, and explain the results of the above
experiments.
It has not been possible to calculate precisely the pH gradient obtained when
hydrochloric acid diffuses into a dilute buffer solution, as in the above situation.
A very rough estimate of the resulting bimalate gradients indicates that they are
sufficient to cause the observed aggregation, but this interpretation must be tentative
until a more exact calculation of the gradients is achieved.
Chemotaxis of bracken spermatozoids 195
An alternative suggestion, which is more complex yet consistent with these
observations, is that sensitivity to the undissociated malic acid molecule, plus an
independent negative chemotaxis to low pH's, might produce this aggregation.
Although there is some indication of negative chemotaxis to low pH's in washed
sperm suspensions, the phenomenon of electrochemical orientation of bracken
spermatozoids (Brokaw, 1957) suggests that the spermatozoids are sensitive to a
charged ion.
Chemotaxis of bracken spermatozoids from a drop containing io"4 M sodium
L-malate and 0-015 M sodium phosphate buffer, pH 7-5, into one containing io~* M
sodium L-malate and 0-015 M sodium phosphate buffer, pH 6-2, has been observed.
The response is comparable to that observed when a drop containing io"3 M sodium
L-malate is placed in contact with one containing io~* M sodium L-malate, both
drops containing the same buffer.
The gradient of the logarithm of concentration is the important variable in fern
sperm chemotaxis (Pfeffer, 1884). Since Fig. 1 shows that the gradient of the
logarithm of the bimalate ion concentration will be nearly equal to the gradient of pH
at pH's above 6, the bimalate hypothesis predicts that bracken spermatozoids should
be as sensitive to a pH gradient as to a malate gradient. This agrees with the above
result, as the gradients set up by the diffusion of sodium phosphates will be of the
same order of magnitude as the gradients set up by the diffusion of sodium malate.
Bracken spermatozoids orientate to a voltage gradient only if the sperm suspen-
sion contains a chemotactically active compound (Brokaw, 1957). The amount of
sodium L-malate which must be added to obtain this electrochemical orientation is
less at pH 6-8 than at pH 8-1 by approximately an order of magnitude. Fig. 1 shows
that the malate concentration required to give a given concentration of bimalate ions
will be twenty times less at pH 6-8 than at pH 8-i. The hypothesis that bimalate
ions are required to sensitize spermatozoids to a voltage gradient is, therefore, in
approximate agreement with the above result.
If the sperm suspension contains sodium maleate instead of sodium L-malate,
addition of a drop of hydrochloric acid leads to the formation of a broader band of
spermatozoids than when malate is used, and when the pH of the sperm suspension
is low (6-2) there is no sharp aggregation. These observations suggest that in this case,
too, the singly ionized ion, bimaleate, is the active species, and are consistent with the
higher pK^ of maleic acid (6-6).
DISCUSSION
Shibata (1911) suggested that malate might have a cis-configuration, since he, and
Pfeffer, found maleate (CM) to be chemotactically active for many pteridophyte
spermatozoids while fumarate (trans) was inactive. However, the malate ion, which
predominates in solutions of pH above 6, is not likely to exist in the ctr-configuration,
while the bimalate ion will have a cu-configuration. Shibata also found that sperma-
tozoids of Equisetium arvense L. and of Salvinia natans Allioni were positively
chemotactic to solutions of dilute acids. He found that the sensitivity to a gradient
of H+ ions was just as strong as the sensitivity to a gradient of malate, and that the
13-2
196 C. J. BROKAW
effects of H+ and malate concentrations were not additive. Since he did not show
that the response to H+ required the presence of malate, he concluded that the
sensitivities to H+ ions and to malate were independent. My experiments demon-
strating the chemotactic interrelationship of malate and pH suggest that these
spermatozoids are sensitive to the bimalate ion and not to the malate ion or the
malic acid molecule, in agreement with all the above observations.
Shibata also studied the spermatozoids of Isoetes japonica A. Braun, which show
positive chemotaxis to gradients of malate, fumarate, and OH~ ions. The above
considerations suggest that in the case of Isoetes spermatozoids, the malate ion,
rather than the bimalate ion, may be active. However, it is difficult to see how a pH
gradient in solution above pH 6 could produce significant gradients of malate ion
(see Fig. 1). It would be interesting to know if Isoetes spermatozoids are attracted
by OH~ ions in the absence of malate.
SUMMARY
1. Bracken spermatozoids are chemotactically attracted by malic acid salts and
in a pH gradient aggregate between pH 3-5 and 5-6.
2. This response to pH occurs only when the sperm suspension contains malate.
3. At higher pH's, the response to an H+ gradient is as strong as the response to
a malate gradient.
4. These observations suggest that bracken spermatozoids are sensitive to the
bimalate ion rather than the malate ion. This is consistent with an earlier suggestion
that chemotactic activity is associated with the nj-configuration, for the bimalate
ion will have this configuration, while the malate ion and the malic acid molecule
will have the franj-configuration.
This work has been carried out during the tenure of a National Science Founda-
tion Predoctoral Fellowship. I am also indebted to Prof. G. Kenner for suggesting
that the configurations of malate in solution might be significant, and to Lord
Rothschild for introducing me to bracken sperm chemotaxis.
REFERENCES
BROKAW, C. J. (1957)- 'Electro-chemical' orientation of bracken spermatozoids. Nature, Lond., 179,
5*5-
PFKFFER, W. (1884). Locomotorische Richtungsbewegungen durch chemische Reize. Unter. Bot.
Irut. Tubingen, 1, 364-482.
ROTHSCHILD, LORD (1956). FertHizatum. London: Methuen.
SHIBATA, K. (1911). Untersuchungen iiber die Chemotaxis des Pteridophyten Spermatozoiden.
Jb. win. Bot. 49, 1-60.


Chemotaxis of Bracken Spermatozoids : The Role of Bimalate Ions

Electro-chemical' orientation of bracken spermatozoids.

Untersuchungen iiber die Chemotaxis des Pteridophyten Spermatozoiden.

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Chemotaxis of Bracken Spermatozoids : The Role of Bimalate Ions

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