The Effect of Dilution on the Activity of Spermatozoa

The first three suggestions can be readily eliminated. If undiluted sperm is centrifuged for about 30 minutes at 2500 revolutions per minute (10 cm. radius), the spermatozoa can be separated from the testicular plasma; the latter separates out as a transparent opalescent fluid comprising from 15 to 20 per cent, of the total suspension. If a small drop of undiluted sperm is added to this plasma, intense activity occurs as in sea-water. That there is no essential change in the nature of the dissolved gases during the period spent in the centrifuge is shown by the fact that no movement occurs in the normal undiluted suspension when the oxygen content is raised or if the CO₂ content is lowered. This experiment shows fairly conclusively that the absence of movement in the normal seminal fluid is not due to the chemical or physical properties of the medium which surrounds the spermatozoa. We are therefore driven to the conclusion that the activating effect of seawater must be due to the process of mechanical dilution whereby each spermatozoon becomes surrounded by an increased free space for movement. If undiluted sperm is examined under a high power, individual spermatozoa can be seen to be moving wherever there is sufficient space for this to occur. The cells are packed together so closely, however, that any appreciable movement is impossible. One of the striking facts established in this paper is that the cells not only do not move when packed together, but that they do not make any effort to move.

A quantitative analysis of the phenomenon is available if we assume that the degree of mechanical activity can be measured by the rate at which the cells absorb oxygen. That changes in the degree of mechanical activity and changes in the rate of oxygen consumption run parallel to each other is shown by the following figures :

In the following experiments, therefore, the activity of the spermatozoa is measured by the rate at which a unit quantity of cells absorbs oxygen.

The relationship between respiratory activity and degree of dilution was determined by means of the following technique. The testes of a ripe Echinus miliaris or E. esculentus were carefully dried with filter paper and were then cut into two pieces, thereby allowing the sperm to flow out into a dry vessel. Some of this thick creamy suspension was drawn into a capillary pipette and a known number of drops (32 or 64) were transferred to a dry respirometer of the differential manometer type. A fixed bulk (6 c.c.) of sea-water containing egg-secretions was then added, and the amount of oxygen consumed in 10 minutes by the uniform suspension was determined. Half the fluid was then withdrawn and replaced by 3 c.c. of sea-water with or without egg-secretions. The respiratory level was again determined and the process repeated until a very dilute suspension had been reached. The necessity for using egg-secretions and for restricting the length of the whole experiment lies in the fact that it is only when egg-secretions are present that the respiratory level of a diluted suspension remains constant for about one to one and a half hours (see Gray, 1927). Control experiments showed that the quantity of egg-secretions present does not exert any differential effect on suspensions of different concentrations if care is taken to use secretions from a fresh and ripe female, and to use the secretions in a relatively concentrated state. At the end of the experiment 50 drops of undiluted sperm from the testis were analysed for nitrogen, and in this way an approximately absolute value was obtained for the concentration of the various suspensions used. As a general rule 50 drops of undiluted sperm contained 17–20 mg. of nitrogen.

Fig. 1 and Table I show clearly that except in very dilute suspensions the respiratory level at the beginning of active life is not simply proportional to the number of spermatozoa present in a particular sample of diluted sperm, but also depends on the degree to which the original testicular suspension has been diluted with sea-water.

The data summarised in Fig. 1 are more clearly understood by reference to Fig. 2, in which the specific activity of 1 mg. of nitrogen equivalent of sperm is given at varying degrees of dilution. It is obvious that the greater the dilution the greater is the initial activity of individual spermatozoa, but as the dilution increases the effect on the activity becomes less marked.

As a confirmatory test of these conclusions a series of suspensions was examined in which the same amount of spermatozoa was diluted by different amounts (3 c.c. and 6 c.c.) of sea-water (Table II).

These results obviously conform with those of the previous series. In very weak suspensions the activity of a spermatozoon is more or less independent of the dilution, but in stronger suspensions the activity is markedly affected by further dilution. That the reduced activity in strong suspensions is not due to lack of oxygen is shown by the fact that the amount of oxygen consumed by 6 c.c. of a suspension containing 25 mg. N equivalents of sperm is actually less than that of the same volume of a suspension containing 5 mg. N equivalents ; also by the fact that very extensive aeration of the stronger suspensions failed to increase the total respiration.

The only apparent interpretation of these facts appears to be that the comparative inactivity of spermatozoa in concentrated suspensions is due to the lack of free space in which an individual spermatozoon can move. A rough calculation indicates that the undiluted sperm must be diluted to about 800 times its own volume in order that each spermatozoon should have a mean free path of movement equal to its own length. The dotted line in Fig. 2 shows that over a considerable range of dilution the specific activity of a spermatozoon is a linear function of the cube root of the volume of sea-water in which it is free to move. In the case of any given suspension, therefore, the total amount of initial activity is proportional to the number of spermatozoa present and to the average free space in which each cell can move.

Fig. 3 illustrates a point of considerable interest, for it shows that in a concentrated suspension not only is the initial degree of activity of each spermatozoon lower than in a more dilute suspension but that the total energy expended is also lower. In other words, the total amount of energy expended during the whole of active life depends on the initial degree of activation which is itself dependent on the dilution, and is not a fixed quantity depending on the whole resources of the cells.

Mutual inhibition of the activity of unicellular organisms is known in the protozoa. When Paramecium forms spontaneous aggregates, the activity of each organism is reduced. How far this is directly due to increase in population density, and how far it is due to a localised change in environment, is unknown. In a dense suspension of spermatozoa it is possible that frequent collisions between the cells lead to mutual inhibition just as the cilia of Paramecium become less active when in contact with a solid surface. Such conditions, however, would not account for the fact that the total energy expended by a dilute suspension of sperms is relatively greater than that of a denser suspension. The behaviour of spermatozoa almost suggests a voluntary phenomenon in which the contractile effort is proportional to the free space in which the organism can move, and which is limited to part of the contractile machine only. Until more is known of the mutual effect of one cell upon its neighbours the phenomenon will remain obscure.¹

It is well known that the fertilising power of spermatozoa is more rapidly lost when the cells are kept in a dilute suspension than in one which is more concentrated. The explanation commonly given to this fact is that in stronger suspensions activity is inhibited by the CO₂ evolved by the spermatozoa, and in this way the cells conserve their energy. The data given above show that this explanation is only partially correct at the best, and that it may be quite erroneous. In the present experiments the respiratory CO₂ was continuously removed, and it is quite clear that at no point in the life of a concentrated suspension is the respiration per unit quantity of sperm equal to that in a dilute suspension.

As explained elsewhere (Gray, 1928) the initial activity of a suspension rapidly falls unless egg-secretions are present, and these experiments show that the effect of high concentration on the length of life is not due to the narcotic effect of accumulating CO₂, but is due to the fact that in a concentrated suspension only a fraction of the energy in the cells is mobilised and only part of the machine undergoes senescence, while the remainder is free for liberation at any subsequent process of dilution. Fig. 4 illustrates the mobilisation of a further supply of energy by a secondary dilution of an active suspension. It may be mentioned that lowering the CO₂ tension by addition of dilute alkali had no effect on the respiratory level immediately prior to the second dilution.

If the conditions of a dilute sperm suspension are such that the CO₂ generated is allowed to accumulate, the rate at which the mobilised energy is expended will decrease as time goes on, and this will also tend to increase the length of life during which the cells are motile when the CO₂ is removed by subsequent dilution. These conditions appear to have existed in some of Cohn’s (1918) experiments, where the suspensions used were sometimes very dilute and where the full initial activity was mechanically possible. In such suspensions it should be possible to show that the initial rate of respiration per cent, of sperm was the same in all cases.

One of the most striking features of this investigation is the high level of respiration reached by moving spermatozoa. One gram of spermatozoa, newly shed into a large bulk of sea-water containing egg-secretions, uses up approximately 3 c.c. of oxygen in 10 minutes at 17 ° C. In a large E. esculentus the testes may contain at least 30 gm. of ripe sperm and this will require about 100 c.c. of oxygen in 10 minutes; this quantity of gas is contained in about 12 litres of sea-water, so that the oxygen tension in the region of a spawning urchin must fall very appreciably and is possibly of some ecological importance.

The facts described also indicate that if sperm (e.g. that of mammals) is required for transportation, it should invariably be kept in an undiluted state since dilution involves activity, and activity is the cause of irreversible senescence (Gray, 1928). Even in the undiluted state, the potential activity of Echinus sperm decreases with age. How far this is due to bacterial contamination is as yet unknown.

• (i) The relative inactivity of the spermatozoa in the undiluted sperm of Echinus is not due to the physical or chemical constitution of their natural medium in the testis, since the cells are intensely active in this medium when the majority of spermatozoa are removed by means of a centrifuge.

• (ii) The total activity of any suspension, as measured by its demand for oxygen, is proportional to the number of spermatozoa present and to the average distance in which each cell is free to move. Inactivity in the testis appears to be due to mechanical overcrowding, each cell appearing to exercise a restraining or allelostatic effect on the activity of its neighbours.

• (iii) The total energy expended during the life of a spermatozoon, as well as the level of activity exhibited immediately after activation, depends on the degree of dilution of the suspension examined.

• (iv) The relatively long life of concentrated suspensions is not due to the narcotic effect of accumulated CO₂, but is the result of an incomplete state of activation on the part of each spermatozoon.