The Chemotaxis of Spermatozoa and its Questioned Occurrence in the Animal Kingdom

A CURIOUS and very interesting position has arisen concerning the question of chemotaxis between animal eggs and spermatozoa. Although many writers have assumed (without experiment it is feared) that the conjunction of egg and spermatozoon in fertilisation is very generally the result of the ovum secreting substances which direct the course of the moving sperms towards it, actual references to original papers show that the foremost workers are in dispute as to the reality of this phenomenon.

It is well known that in plants a chemotaxis of the above type can be easily demonstrated, the best example being the attraction of malic acid for fern antherozoids, and the directive effect which is supposed to be exerted by this substance in the mucus of the archegonia. The classic researches into the phenomenon were made by Pfeiffer, who demonstrated chemotaxis by using capillary tubes filled with the substances to be tested. These researches, however, were confined to the plant kingdom.

Loeb on more than one occasion has refused to accept the theory of chemotropism in connection with animal eggs. In his Dynamics of Living Matter (p. 153, New York, 1906) he states:— “ The appearance of Pfeiffer’s paper aroused in many the hope that it might be shown that the animal egg, too, attracted the spermatozoon in some such chemotropic or chemotactic way; but all the experiments thus far made in this direction by J. Dewitz, Buller, and others— I have made quite a few experiments myself on this subject—have without exception shown that such is not the case in the eggs thus far tried.”

This is a very definite statement and it has undoubtedly influenced other writers. [Loeb still holds to the above (see The Organism as a Whole, p. 93, New York, 1916)]. Thus Kellicott in his work on General Embryology doubts whether chemotaxis is present in the animal kingdom at all.

On the other hand Frank R. Lillie, in that excellent little book The Problems of Fertilisation (Chicago, 1919), insistently refutes the doctrines of Loeb and adheres to the belief that there is at least a certain amount of chemotaxis. He concludes a chapter dealing with this problem with the following guarded statement:— “ The meeting of the egg and the spermatozoon is to be regarded neither as a matter of random activity of the spermatozoon alone, as some have been inclined to regard it, nor yet exclusively as a result of direct orientation of the spermatozoon towards the egg by chemotaxis, as others have supposed. It appears really to be a more complex event in which the various forms of behaviour of the spermatozoon may all play a part, as indicated.”

In view of this contradiction of opinion it is necessary to look into the experiments which have been carried out, and particularly into those referred to by Loeb, i.e. those of Buller, for example.

Buller (a botanist familiar with the work of Pfeiffer) conducted an extensive and apparently very careful series of experiments on Echinoderms at Naples in 1901. He commences his paper (Q.J.M.S., 1902) by referring to the work of the botanists and then, after mentioning zoologists who had already assumed chemotaxis in the animal kingdom, he writes :—” At present to the best of my knowledge not a single case is known where chemotaxis plays a rôle in the fertilisation of the eggs of animals.” Buller used the Pfeiffer method with capillary tubes and investigated eleven species of Echinoderms. The result was that no attraction of the spermatozoa into a tube could be observed. Substances other than egg extracts were used but with no more success. The sperms went into and out of the tubes containing egg water with indifference. Buller’s work appears thorough and it is not surprising that Loeb has made a stand on the result, especially as his own capillary tube work was equally negative.

However, even Lillie, who opposes Loeb’s view, does not seem to have had much success with capillary tubes and, adopting another method of showing chemotaxis, criticises the tube method in a way which is undeserved. Yet one worker has obtained a different result, for J. de Meyer using methods exactly similar to those of Buller obtained plugs of sperms in his tubes. Lillie adds in regard to this:—” He thus disagrees entirely with Buller; but as neither author gives any quantitative data, it is difficult to find the cause for the disagreement.”

It will be seen from this preamble that there is some importance in looking into the question. The results of Buller and Loeb on the Echinoderms have been used as the basis for rather broad generalisations—indeed for the statement that chemotaxis between eggs and sperms does not occur in the animal kingdom. It was on these grounds that we set out to investigate the conditions in the same group of animals, the Echinodermata, using in particular the method of capillary tubes which is undoubtedly responsible for the present confusion.

The first experiments made by us were carried out using a modification of the well-known method of counting blood corpuscles. As the method may be employed for the investigation of heliotropism, it will be described briefly here. The animal used for the experiments was Echinus esculentus, of which any number of specimens could easily be obtained at low tide at Port Erin, Isle of Man. Ripe males and females were sought (the breeding season occurs in the spring months), and suspensions of spermatozoa of various concentration were made by breaking up the ripe testes into sea-water and filtering the coarse tissue through fine silk. The ripe ovaries were then extracted from a female and the eggs placed in bags of fine bolting silk (carefully washed) of such a grade that ova of Echinus esculentus did not easily pass through the meshes. These bags were carefully suspended in dishes of sperm suspension. It was thought that any secretions or extracts from the eggs diffusing out into the sperm suspension might by chemotaxis raise the concentration of the spermatozoa in the proximity of the silk bags. Such an unequal sperm distribution could be measured by actual counts. Counts were made using a Zeiss Thoma hæmacytometer slide together with long fine pipettes. The technique is very similar to that involved in bacterial counts when vaccines are being standardised. A definite length of sperm suspension is drawn into the pipette and a mark made on the tube (or the mark is made first). It is then possible to dilute this fluid to any appropriate extent by sucking up one or more similar lengths of dilute formaline sea-water, allowing a little air to enter and separate each length. The fluids sucked up are next expressed on to a hollow slide, and resucked up and down so that complete mixing takes place. After this procedure a drop or two is inserted on the counting slide and the usual technique followed.

The results of a series of observations made in this way were, however, unreliable, owing largely to the tendency for sperms to adhere for a time in clumps. We discontinued the method temporarily in order to follow out our plans with capillary tubes.

The capillary tubes used were almost all of the same diameter, viz., 0.5 to 0.7 mm., and in any one experiment the diameter of all tubes compared was the same. The length was about 3 cm. A few tubes were only 0.3 mm. and others about 1 mm. in diameter.

Egg-water was made by breaking up small parts of a ripe ovary in 20 to 40 c.c. of sea-water and filtering the suspension through well - washed (with sea - water) filter paper. The capillary tubes, sealed at one end, were then filled as rapidly as possible by means of a good air pump.

Other tubes were filled as controls, with sea-water, with filtered cœlomic fluid from the male and female sea-urchins, and with other modified solutions. The hydrogen-ion con centration was usually determined. After the tubes had been left side by side in the sperm suspensions for a given time, each tube was picked up with forceps and the outside rapidly wiped with filter paper moistened with sea-water. The air being at a low temperature in the Port Erin Station, there was no risk of losing any of the contents of the capillary tube as the result of expansion. The tubes were next placed together in sea-water and compared side by side under the microscope.

As any directive action of the contents of the capillary tubes would be related to the diffusion of the contents into the sperm suspension, a few experiments were made to gain a little information on this point. A series of tubes was taken ranging in diameter from 0.3 mm. to 1.0 mm., and these were filled with solutions or suspensions of alum carmine in sea-water. They were placed in clean sea-water and the diffusion of the colour outwards was watched under a lens. At the end of five minutes nothing was observed from the finest tubes, but those of the diameter usually employed in the experiments gave little coloured areas at their mouths. After the first ten minutes this area did not seem to expand, and at the end of twenty-four hours these tubes were still as deeply coloured as at the beginning of the experiment except near the open end. In the larger tubes rarely used in the experiments the diffusion was greater and the fluid was only pink near the closed end. A satisfactory gradient seems therefore to be set up in the tubes, assuming that the rate of diffusion of the egg-water is somewhat similar.

One other feature of importance was the thigmotactic effect of the glass tubes. A certain number of sperms always adhered to the glass. The effect of the glass should, however, be approximately the same in the tubes compared, and allowance has been made for this disturbing effect.

The first experiments made at Port Erin, Isle of Man, with the capillary tubes gave a decided positive result, rather to our surprise after reading Buller’s work. Each tube containing egg-water was almost plugged near the mouth with spermatozoa, the number in the other tubes being much smaller. A most interesting feature, however, was the presence of motile sperms farther up the egg-water tubes than up those containing normal sea-water. Our confidence in these results was strengthened by the fact that tubes containing egg-water prepared in the same way from Echinocardium cordatum (ripe ovaries) had no more effect on the sperms of Echinus esculentus than had ordinary sea-water. (The P_H of both egg waters was the same.) Thus, whatever the explanation, the actual results are absolutely contrary to those obtained by Buller on other sea-urchin material.

When the results came to be written up in Liverpool, it was realised that, although very definite, the experiments had not been carried out in as great detail as was desirable, and the authors felt that it was not by any means certain that spermatozoa had actually been attracted (directed is the better term) into the egg tubes. It was noted that many sperms within the tubes were non-motile, and since it was known that egg extracts of a certain concentration cause sperms to lose their motility and aggregate, it was thought possible that the difference in appearance between the egg tubes and the controls might be explained in another way. The sperms are moving actively in all directions in the sperm suspensions. They would consequently move into all the capillary tubes immersed in them (as is actually the case). Now it is quite conceivable that they move out of, as well as up, the sea-water tubes, so that the concentration in these does not exceed that in the suspension outside, whilst they are immobilised within the egg-water tube and remain there to accumulate gradually.

In other words, the accumulation in the egg-water tube might be due to a “holding” of all sperms which entered the tube, and not to an actual directive effect at all.

To gain further light on the phenomenon other experiments were set going, and with satisfactory results, although it must be admitted that the above problem is not an easy question to settle. At the outset, however, it should be made clear that whether or not the egg secretions exert a real directive action on the sperms, the tube experiments carried out by us give results quite different from those of Buller and Loeb.

It was too late to work with sea-urchins at Port Erin, but fortunately farther south, Echinus esculentus could be obtained with ripe gonads at Plymouth during May and June. The following experiments were carried out at the Marine Biological Station at Plymouth, and the authors are very grateful to the kind assistance accorded them by Dr Allen and his colleagues at the Laboratory.

Six dishes were used with a similar sperm suspension in each. Each dish contained four tubes, two (the controls) with sea-water, the other two filled with egg-water. This gave twelve pairs of tubes for the comparative tests.

At the completion of the above experiment each pair of tubes was removed to normal sea-water and re-examined after three and a quarter hours. It was noticed that the sperms in the egg-water tubes were still very active and were moving up to the end of the tubes; the block at entrance had been some what dispersed. Sperms in sea-water tubes had commenced to lose motility.

For the first fifteen minutes or so after immersion there was no great difference between the two capillary tubes, the egg-water tube, and its control. Sperms were passing into both. It then became apparent that the egg-water tube was collecting more than the control sea-water, and at the end of one and a half hours there was an obvious excess of sperms in the egg-water tube.

The length traversed by the sperms in this experiment was always greater in the egg-water tubes. After one and a half hours, however, it was difficult to make a comparison of the numbers of sperms which had entered. The general impression given by the egg-water tubes of the third, fourth, and fifth pairs was that a greater number of sperms were actually directed (not merely captured — otherwise they would have passed up a greater length in the control sea-water tube than the egg-water tube).

In the pairs of tubes left longest in the sperm suspension, i.e. 9, 10, 11, and 12, the sperms had practically attained the top of both tubes, but the blocks at entrances were disturbing influences.

Fig. 1 is a sketch of the egg-water capillary tube and the control tube at the end of an experiment not unduly prolonged (Nos. 3, 4, and 5 in Experiment I., for example). The effect in the egg-water tube is fairly typical. There are three regions to be distinguished. Near the mouth the distribution of sperms is dense and all the sperms are active. Some are aggregated in spots on the inner wall of the tube. Following this region there is a densely crowded section of the tube which can be seen with the naked eye. Finally, we come to the region farther away from the mouth where the sperms are approximately evenly distributed and much less dense. The numbers here are similar to those in the upper portions of the control tubes.

Twelve Petri dishes containing sperm suspension were taken, in each of which three capillary tubes were deposited — one contained sea-water, one cœlomic fluid, and the other contained sea-water filtered off from mature eggs.

The result is a confirmation of Experiment I.

Seven dishes were used containing sperm suspension of the same concentration. Each dish was supplied with three capillary tubes which may be called a, b, and c tubes. The a tubes contained sea-water; b tubes contained egg-water, i.e. sea-water which had been filtered off unbroken pieces of ovary; and the c tubes contained sea-water filtered from crushed ovaries.

A greater number of sperms were found in the capillary tubes containing sea-water which had been in contact with the eggs, and (neglecting small variations) the length traversed by motile sperms was greater in the egg-water tubes than in the sea-water controls.

Four dishes were used for the experiment, each containing three capillary tubes. The same concentration of sperm suspension was placed in each dish. The capillary tubes of each set contained: (a) sea-water, as control; (b) sea-water + slight trace of HCl; (c) egg-water.

The HCl was added to sea-water in order to reduce the motility of the sperms after they had entered the capillary tubes containing the mixture. The idea was to try and prevent those entering from moving out again, in order to find out whether an accumulation of sperm could be produced in this way. (It must not be forgotten, of course, that the addition of HCl might prevent sperms entering the tubes— this would again, however, imply a chemotaxis, although of a negative type.)

There was no question about the action of the HCL The reagent affected the motility of sperms in tubes b, but it did not seem to prevent them from entering. It did not, however, result in anything like the accumulation of sperm that was found in the egg-water, and in every case the egg-water tubes contained a great excess when compared with the control.

In this experiment ten dishes were used and the concentration of sperm suspension was greater in each succeeding one. It was found impossible to measure the concentration in actual number of sperms per c.c. at the time of the experiment, and it appeared as of little real importance. The concentrations varied from dish I, containing sea-water that was merely opalescent with sperm, up to dish 10, in which the sea-water was quite milky. In each dish there were two sets of three capillary tubes (giving twenty comparisons).

The capillary tubes may be termed x, y, and z.

• y contained normal sea-water as usual control.

• z contained sea-water + KCN.

• x contained egg-water as usual.

Note. — As it was not possible to examine all the tubes at the same instant, the order of examination of dishes was varied, as will be seen from the table, in order that results might be obtained from dishes of dense and dilute sperm suspension after approximately the same period of time.

Note. — The KCN solution was tested in the following way : — 10 c.c. of dense sperm suspension were placed in each of several watch glasses. Varying amounts of a 1 per cent. solution of KCN in sea-water were then added. Five drops resulted in a cessation of motility within the concentrated region in about eight minutes, but sperms were still active in adjacent regions. Ten drops of KCN resulted in many sperms losing their activity at once; all motility was absent after two minutes. The KCN capillary tubes were filled with a solution made up of 10 c.c. sea-water to which 5 to 7 drops of 1 per cent. KCN had been added.

This prolonged experiment serves to show that the natural conditions of the egg-water tube are not reproduced merely by adding an immobilising reagent to sea-water. It would appear again, therefore, that the sperms in the egg-water tube are actually directed there. The majority of tubes show this, although it will be noted that one or two unexplained differences occur as in most other sets of experiments, and the results with tubes of abnormal diameter are somewhat different as might be expected.

In this experiment seven dishes were used, each containing eight tubes. The concentration of sperm suspension in dish 1 was very dense (quite milky). In dishes 2, 3, and 4 it was about two-thirds the density of dish 1, and in dishes 5, 6, and 7, one-quarter the density of dish 1.

The tubes were as follows : —

Note. — The acid solutions of varying P_H were prepared by the addition of small amounts of a special fluid containing HCl, Tiedman’s sea-salt, sodium carbonate, and sea-water.

The results of the comparisons are stated in each column by the order in which the tubes are mentioned, commencing with that of the maximum reading. Tubes bracketed together are similar in regard to the reaction.

The egg-water tubes were again conspicuously filled with spermatozoa. In regard to the other tubes it would appear that on the whole any alteration of the P_H of the sea-water resulted in the entrance of fewer sperms than in normal sea-water. Where this was not the case the alteration of the P_H had been insufficient to make any appreciable difference. It also appeared as if a considerable reduction of P_H (as in tubes e) was not so disturbing as an increase to 8.8 and 8.6 (tubes f and g).

The above experiment (No. VI.) was repeated, the confirmed the P_H ranging from 8.2 to 7.0. The results above confirmed the above.

A series of experiments similar to those with Echinus esculentus was carried out with the reproductive products of Teredo navalis which spawned in dishes in the laboratory. A fertilisation control gave 100 per cent. eggs fertilisable. Seven dishes were used each containing one capillary tube of sea-water and two of egg-water (each containing egg exudations from different females A and B).

Only two cases give a positive result in favour of the egg-water. As one always finds one or two varying tubes it must be concluded that there is no directive action, at least as compared with that of Echinus esculentus egg-water. Unfortunately it was not possible to continue the experiments with Teredo to the same extent as those with Echinus esculentus.

Six dishes were used with the same concentration of sperm suspension. Five capillary tubes were placed in each dish, the contents being as follows: —

• a —Egg-water.

• b —Sea-water control.

• c, d, and e —Sea-water + a few granules of quinine bihydrochloride reducing the P_H of the mixtures to 6.6, 5.9, and 4.6 respectively.

The experiment was conducted on similar lines to those with HCl and KCN, an attempt being made to reproduce the conditions in the egg-water tube on an assumption that the sperms might be accumulating therein through capture (immobilisation).

There were again without any doubt more sperms in the egg-water tubes than in the controls or in any of the others. The experiment showed, however, that it is possible to cause an increase of the sperm number in a capillary tube as a result of capture by immobilisation. Probably both attraction and capture are taking place in the egg-water tubes.

In Experiment X. four capillary tubes were used in each of four dishes. The sperm concentration varied in the different dishes, the greatest concentration being in No. 1. The capillary tubes contained : —

a —Sea-water as control.

b —Egg-water (P_H 8.0).

c —Egg-water + HCl (P_H 4.8).

d —HCI and sea-water (P_H 2.6).

Experiment XI. was a repetition of Experiment X., but with dense suspensions of sperm.

These experiments were very definite. It is evident that egg-water results in a great increase of sperms in the capillary tube as compared with the capillary tube of normal sea-water. The addition of HCl, however, to egg-water increases the number still further, possibly by adding the effects of capture to those of chemotaxis. Similarly in some cases, but not in all, the addition of HCl to sea-water alone resulted in a greater number of sperms being in the tube than in the tube containing sea-water alone. As was to be expected from the immobilising action of HCl, a smaller length of tube was traversed by the sperms in the case of the HCl sea-water than in any of the others. The addition of HCl to egg-water reduced the activity so far as length of tube traversed was a measure, but it was still greater than in the tube containing sea-water only.

This experiment was repeated for the purpose of photographing the tubes. The results (five tests) were the same as before and are shown in the illustration fig. 2. The differences in the types of sperm collections in the different tubes is rather interesting.

Experiment X. —Photograph of the capillary tubes A, B, C, and D after immersion in sperm suspension.

A, contained sea-water.

B, contained egg-water.

C, contained egg-water + HCI.

D, contained sea-water + HCI.

In the control tube A the number of sperms is small and there are no dense areas. In the egg-water tube B there are scattered clumps of sperms near the mouth of the tube (just visible in the photograph), whilst higher up there is a dense mass forming a “plug.” Above this many motile sperms are found. In tube C the numbers of spermatozoa are greater still and a very obvious “plug” is seen. Tube D contained comparatively few spermatozoa. The photograph does not show the scattered moving spermatozoa. The difference between tubes A and B is typical of that which constantly occurred throughout these experiments.