Effects of Infra-Red Light on Subsequent Cleavage of the Eggs of Urechis Caupo

Eggs were secured from animals recently collected and in good condition. These eggs were suspended in sea water and allowed to settle in a single layer at the bottom of a specially constructed glass dish. The design of this dish was such that control eggs could be kept apart from those irradiated, and yet be in the same body of sea water. The dish was fastened in such a position that the eggs in the section to be irradiated were directly over the slit of the quartz monochromator. The monochromator and source were those previously described (Nelson and Brooks, 1933). Most of the radiation used lay between 0·7 and 2·0 μ, and was nearly plane polarised.

Different samples of eggs were irradiated for periods of from 15 to 60 min. After irradiation both exposed and control eggs were divided into two or more portions, one of each being inseminated as soon as possible in most cases, and the other portions inseminated after periods varying from 30 to 435 min. Freshly collected sperm was used for each insemination so as to avoid all danger of staling of the sperm. As a further safeguard, all steps of these experiments were carried on in a room whose temperature was kept at 1–10° C., i.e. at about the temperature normal for these animals. This low temperature is probably the principal factor responsible for the higher proportion of successful fertilisations obtained in these as compared with our previous experiments which were carried out at ordinary room temperature.

After insemination the small dishes containing the fertilised eggs were all immersed in the same large vessel of sea water, where each lot of eggs rested on the bottom of its own small dish, but in free communication with the larger mass of sea water. This procedure may also have contributed to the higher proportion of eggs forming fertilisation membranes in the present experiments : eggs unfertilisable immediately after irradiation may have recovered sufficiently to be fertilised by the viable sperm which were present in the larger body of sea water during most if not the whole duration of development.

After insemination, periods varying from 25 to 815 min. were allowed for development, and then individual lots of eggs were removed and classified into groups including those which had attained the following stages : fertilisation membrane but no cleavage, 2 cells, 4 cells, and 8 or more cells; the number of unfertilised eggs was also determined.

The number of eggs examined in each such lot varied in the case of the irradiated eggs from 297 to 697, the average being 425. The corresponding figures for the nonirradiated controls were 322 to 704, and 480. Other experimental details are given in the tables.

Under the more favourable conditions of the present experiments 15 min. irradiation was not enough to inhibit membrane formation. In Exp. I eggs were irradiated for this length of time and divided into three lots which were inseminated 5, 33, and 63 min. after irradiation, and observed about an hour later. In all, 2316 irradiated eggs and 1974 control eggs were counted. In none of the three cases was there any significant effect, the fraction successfully fertilised varying irregularly between 98·6 and 100 per cent, for both irradiated and control groups.

Irradiation for 30 min. considerably reduced the fraction of eggs forming fertilisation membranes in one experiment (Table I), but even longer exposures (35–60 min.) failed to produce this effect in four subsequent experiments, three of which are given in Tables II and III. The reason for this difference is not apparent ; the interval elapsing between irradiation and insemination is not a factor.

The cleavage of eggs which had formed fertilisation membranes was definitely affected by irradiation in all cases where data are available. This is shown particularly well by Exp. IVa, b, c, in which the only factor varied was the interval between fertilisation and observation. The irradiated samples in all cases had fewer eggs in advanced cleavage stages, and more in the earlier stages or uncleaved state than did the corresponding controls.

The eggs appear to recover from the effects of irradiation both before and after fertilisation. The occurrence of this apparent recovery during the interval preceding insemination is indicated by the smaller differences between irradiated and control eggs in Exps. IIIa or Va, as compared with IIIb or Vb (Table II), or in Exp. IV d as compared with IVb, or IVc (Table III). In all these cases the time allowed for development after insemination was also varied, but, regardless of whether the total time between irradiation and observation was longer or shorter, increasing the interval between irradiation and insemination resulted in a decrease in the difference between the irradiated and control lots. A similar process appears also to have gone on after insemination as shown by Exp. IVa, b, c, in which progressively longer intervals were allowed for post-fertilisation development.

These experiments show that infra-red light lying in the general region of 0·7–2·0 μ produces partial inhibition of cleavage of fertilised eggs of Urechis caupo. Under certain conditions, which are not clearly defined, it also decreases the fraction of the eggs which form fertilisation membranes upon insemination. Both before and after insemination there is convergence in the behaviour of irradiated and control eggs; this is most conspicuous before insemination. It is natural to interpret this as due to recovery from the effects of irradiation, but it is possible that non-irradiated eggs undergo some sort of deterioration or staling, and that this process is partially inhibited by infra-red irradiation. In this connection it is of interest to recall Loeb’s observation that mature unfertilised eggs of Asterina and Asterias normally cytolyse within a few hours after shedding unless oxidation is prevented (Loeb, 1902, 1905). Irradiation might conceivably interfere with similar deleterious oxidations in Urechis eggs as well as have the initial effect of decreasing the possibility of fertilisation and the rate of subsequent cleavage.

It seems apparent that the process, whether it be recovery or delay in staling, is more important before fertilisation than after, because Exp. IVc shows more difference between irradiated and control lots 830 min. after irradiation than does Exp. IVd 690 min. after; the former was inseminated only 15 min. and the latter 435 min. after irradiation.

Finally, it is to be emphasised that none of these effects are due to heating; there was no measurable difference in temperature either between the media in which the exposed and control eggs lay during irradiation, or between the interior of the irradiated eggs and their surrounding medium. The evidence bearing on this point was presented in our first paper (Nelson and Brooks, 1933). The effects seem, therefore, to be photochemical in nature.

In all previous experiments known to us no effort has been made to show that infra-red light has any effect other than by means of generalised heating of the irradiated cells or tissues. In some cases this is obviously the nature of the effect, and in no case is the effect clearly photochemical.

The following explanation may make more clear the distinction between thermal and photochemical effects as here intended. We may suppose that the effect of the light is partially to destroy a substance essential to cleavage, or, to produce a substance which inhibits cleavage. In either case we are concerned with the acceleration of a chemical reaction. This might be accomplished thermally, all cellular reactions being non-selectively accelerated, or photochemically, when only one particular reaction is affected.

Ordinarily, the term photochemical might be taken to imply the absorption of quanta by molecules, the excitation being in a degree of freedom which normally is in the zero energy state, or at least has so little energy that this may be neglected in comparison with the quantum absorbed. There would then be no measurable reaction of molecules except on absorption of a quantum of the incident radiant energy. At the opposite extreme we have dark reactions in which a certain fraction of the reacting molecules have at ordinary temperatures enough energy in the proper degree of freedom to react. Warming the system, whether by convective heat, by radiant energy which has been absorbed and degraded, or by any other means, simply increases the mean energy in all or several of the possible degrees of freedom. It thus increases the probability that any given molecule will have threshold energy for reaction, and so accelerates a reaction which would otherwise still be perceptible.

The present case is intermediate with respect to the size of the quantum, and we have no direct evidence as to whether or not the reaction or reactions accelerated by infra-red light proceed at an appreciable rate in the dark also. We can, however, feel reasonably certain that the infra-red quanta produce their effects before being dissipated as quanta of magnitudes corresponding to the average thermal energy per degree of freedom in the system, i.e. degradation from about 2 × 10⁻¹² to about 2 × 10⁻¹⁴ ergs. Quite possibly the absorption of a single quantum leads to reaction in the case of many or most molecules, some degree of thermal preactivation being perhaps requisite.

Other possibilities might be suggested, but it is manifestly impossible to discuss all the ways in which the absorption of infra-red light might affect cleavage.

1. Eggs of Urechis caupo were exposed to infra-red light of wave-lengths from 0·7 to 2·0 μ.

2. Upon subsequent insemination the rate of cleavage and sometimes also the proportion of the eggs forming fertilisation membranes was reduced.

3. This effect gradually disappeared after irradiation, especially during the time preceding insemination. This has been interpreted as due either to recovery from the effects of the irradiation or to a more lasting retardation of normal ageing processes in the eggs.

4. The photochemical nature of these effects is pointed out and discussed.

This research was based upon preliminary work made possible by a grant from the Committee on the Effects of Radiation upon Organisms of the National Research Council, and was further supported by grants from the Board of Research of the University of California. These are gratefully acknowledged.