The investigations to be described in this paper were originally undertaken in order to throw light on the utilisation of protein as a source of energy by the developing selachian embryo. It has been shown that in the case of the chick (Needham (24), Fiske and Boyden (14)) and of the frog (Bialascewicz and Mincovna (4)) there exists a peak in the intensity of protein breakdown during embryonic life, on each side of which the catabolism of these substances is much diminished. In the case of the mammal (cow and sheep, Lindsay (23) and others) the descending limb of the curve has alone been observed. Our intention was to extend these investigations to a selachian egg, and we obtained several interesting results, although special difficulties were encountered, e.g. (a) the lack of any good series of weighings of selachian embryos, and (b) the remarkably high concentration of urea in elasmobranch tissues, which makes it difficult to regard it wholly as a waste-product.

Experiments by Denis (8) in 1912 showed that in selachians urea is the principal form in which nitrogen is excreted. Working on Mustelus canis, she found that its urinary urea formed 84·7 per cent, of the total waste nitrogen, with ammonia 5·1 per cent, and uric acid 0·5 per cent.

We therefore set out to determine the urea and ammonia content of selachian eggs at all stages during their development. The method used was a modification of that described by Folin (15) for blood-urea. The contents of the eggs was ground in a mortar with sand to as uniform a pulp as possible, then diluted, and the proteins precipitated by boiling with acetic acid at pH just green to brom-cresol-purple. Owing to the presence of fats and lipoids in the yolk which were not carried down on the precipitate of protein, centrifuging had often to take the place of filtration at this point, but we were usually able to prepare a clear or slightly opalescent solution for the urea and ammonia estimation. This is accomplished in Folin’s method by decomposing the urea with soya-bean urease acting at 50° for half-an-hour in the presence of a few drops of phosphate buffer at pH 7·0, and then distilling over the ammonia rapidly into ice-cold 0·05 N hydrochloric acid after adding a saturated solution of borax to the urease-urea mixture. The ammonia in the distillate is estimated colorimetrically by Nesslerisation. Naturally each estimation must be accompanied by a duplicate without urease in order to know the amount of preformed ammonia present, and this value must be deducted from the other.

The eggs used were those of the “rough-dog,” Scyllium canicula, with a few of Pristiurus melanostoma: the results obtained are given in Table I.

Table I.
graphic
graphic

The determinations were made on one egg only in each case unless two or three sets of figures are given beside one length ; this indicates that more than one egg of that embryo-length was analysed. It will be seen that only in very few cases were we able to obtain more than one embryo of a given age, and the individual estimations necessitated by this fact are the cause of the considerable scattering of the observations (see Fig. 1). After the embryo had lifted itself off from the yolk and was simply attached to it by its umbilical cord, it was not difficult to determine its age by measuring its length, but in the earliest stages, while segmentation was proceeding and the blasto-derm was enveloping the yolk, the determination of age presented more difficulty, and we simply noted the diameter of the orange-coloured blasto-derm standing out against the bluish yolk.

As the work proceeded we were surprised to find comparatively large amounts of urea present in the earliest stages. The question was un-fortunately confused by the fact that the only egg we were able to obtain which showed absolutely no trace of development gave an exceedingly low result. In our opinion, however, a certain amount of urea is present in the undeveloped egg, before the embryo has embarked on any appreciable metabolism of its own, and the urea which it forms and excretes during its development is only a contribution to a stock already provided by the maternal organism. These considerations are illustrated by Fig. 1, which shows the data graphically. The points for the stages before the embryo has a measurable length are all grouped along the ordinate.

As Fig. 1 shows, there is undoubtedly a production of urea by the developing dogfish embryo, and although owing to the reason already given the scattering of points is considerable, they seem to fall on each side of a curve very slightly concave to the abscissa and approaching a straight line during the latter part of its course. This line begins at a point corresponding to about 4·6 mg. of urea nitrogen in the undeveloped egg and attains some 20 mg. of urea nitrogen at the time of hatching. It is probable, then, that each Scyllium egg is supplied by the maternal organism with this initial amount of urea, and that the catabolism of the embryo adds to it as development proceeds. The horizontal line on Fig. 1 illustrates diagrammatically the maternal contribution. As for the ammonia-content of the egg, it seems to re-main relatively constant (except for one or two high values) at 0-5 mg. per embryo, with perhaps a slight falling tendency.

The accessory scale on the right-hand side of Fig 1 measures off in mg. the contribution of the embryo to the urea of the egg, and the result is shown in Table II. Unfortunately no good series of weighings exists for Scyllium embryos, but, as is well known (Fulton (17), Kearney (21)), the length of fish embryos increases at first far more rapidly than the weight. Now this evidently implies that when the points are plotted against weight, the slight concavity towards the abscissa shown in Fig. 1 will be greatly accentuated, because the distance between any two points will be drawn out in the later stages and compressed in the earlier ones. This will happen because equal increments of length mean much greater increments of weight in the later stages than in the earlier. Plotted against weight, then, the urea-content curve would rise sharply to a certain point and then very slowly. Accordingly, when the urea produced by the embryo is referred to 100 units of embryo weight, a peaked curve would result.

Table II.
graphic
graphic
Table III.
graphic
graphic
Table IV.
graphic
graphic
Table V.
graphic
graphic
Table VI.
graphic
graphic
Table VII.
graphic
graphic
Table VIII.
graphic
graphic

This expectation we found to be fulfilled as far as possible when we used the figures of Kearney (21) for Mustelus canis as weight data. We lay no emphasis on the result, but it will be admitted that the shape of the ascending weight-time curve for Mustelus probably does not differ much from that of the related Scyllium. As the last column of Table II shows, over the range covered by Kearney (2·5 to 8·0 cm.) a descending curve is obtained, and the dogfish may therefore be said to be in the same position as regards its protein utilisation as the mammal, i.e. a curve has been obtained which is probably the descending limb of a peak.

The presence of urea in the eggs of elasmobranchs is not reported here for the first time. As is well known these fishes have a very special relation to this substance. In 1858 Stadeler and Frerichs(34) isolated “kolossale quantitäten von Hamstoff” from the organs of plagiostomes, obtaining a solid mass of urea nitrate when they added nitric acid to their final concentrates. One liver of an adult Scyllium canicula gave them 2 oz. of urea, and similar high figures were reported for Acanthias vulgaris. Teleostean fishes, however, and the cyclostome, Petromyzon planeri, yielded practically no urea, at any rate not more than would be present in mammalian tissues. Stadeler (33) confirmed the selachian results on Raia batis and clavata and on Torpedo marmorata and ocellata. In 1861 Schulze (30) repeated and confirmed Stadeler’s work on Torpedo, and in 1888 Krukenberg (22) published an extensive work on the subject, in which he related his unsuccessful attempts to demonstrate urea in the bodies of Teleosts (Lophius piscatorius, Conger vulgaris, Aci-penser sturio), a Cyclostome (Petromyzon fluviatilis and Ammocoetes) and a Cephalo-chordate (Amphioxus lanceolatus), although he found large amounts of it in the bodies of Elasmobranch Fishes (Scyllium stellare, Mustelus vulgaris and laevis, Acanthias vulgaris, Squatina angelus, Torpedo marmorata, Myliobatis aquila) and in the Holocephali, Chimaera monstrosa. Particularly interesting were his experiments with eggs—he isolated considerable amounts of urea from a 5 cm. embryo of Mustelus laevis, and from the yolk of Scyllium stellare and Myliobatis aquila eggs, but he could find none in their surrounding jelly or “white.” An egg of Pristis antiquorum yielded 3920 mg. per cent, (wet weight) and a Torpedo ocellata egg 1740 mg. per cent. An Acanthias vulgaris embryo, 17 cm. long, had 3360 mg. per cent, in its muscles, 1800 mg. per cent, in its liver, and 2640 mg. per cent, in its unused yolk. Other work on urea in selachians was done by Gréhant(19) and by Rabuteau and Papillon (27).

More light, however, was thrown on the reasons for this richness in urea when in 1897 Bottazzi (6), working on the osmotic pressure of fish blood, found that elasmobranchs differed fundamentally from teleosts in being isotonic with the seawater.

Bottazzi observed that the selachian osmotic pressure would correspond to some 3·9 per cent. NaCl, but did not emphasise the fact that selachian blood did not contain anything like so much ash. It was left for Rodier(28) to show that the difference was made up almost wholly by urea. Duval (11) has since found that the salts alone would cover only an osmotic pressure of Δ — 1·06°. “High blood-urea,” as Smith(32) says, “is a phyletic character of the orders Selachii and Batoidei,” and its osmotic function was well shown by the reciprocal relation between salts and urea which Smith found to hold in all selachian tissues and fluids.

Whence comes all this urea? In 1901 von Schröder (29) extirpated the livers of a number of fishes (Scyllium canicula) and observed only a small reduction of the urea-content of the muscles, which was 1950 mg. per cent, before and i860 mg. per cent, afterwards. The liver can therefore not be the main source, and possibly all the tissues have the power of forming urea from the amino-groups in the food. Arginase appears to be found in great quantities in elasmobranchs, thus Hunter and Dauphineefao) found the following typical figures:

The arginase of the dogfish liver was twice as active as the most active teleostean liver arginase (in the herring, Clupea pallasii) and forty times as active as the feeblest (in the tommy-cod, Hexagrammos stelleri). It is probable, however, that the contribution of urea made by arginase to the total urea-content of the elasmobranch cells, would not be large. Hunter and Dauphinee made the interesting observation that the undeveloped eggs of Squalus sucklii contained notable amounts of urea, but no arginase, while both were present in an embryo of 20·5 cm.

It may be added that Baglioni (1, 2) found that the selachian heart could not beat properly unless a certain amount of urea was added to the perfusion fluid.

The excretion of urea in selachians does not appear to take place wholly through the kidneys. Denis (9) found that only 20 to 50 mg. were excreted through the kidneys of an adult dogfish per kilo per day. The gills have been found by Duval and Portier (12, 13) to be absolutely impermeable to urea. But van Slyke and White (31) found that large amounts of urea were contained in the bile (up to 72·3 per cent, of the total biliary nitrogen), so that the intestinal tract is probably concerned together with the liver-cells in regulating the urea-content. The kidney does not seem to do much regulation, as Denis (10) found the blood-urea to be uninfluenced by experimental nephritis induced by uranium nitrate or potassium chromate. Another mode of elimination of urea from the elasmobranch body may be through the peritoneal pores, which Smith (32) believes to have an excretory function, for the peritoneal fluid contains 680 mg. per cent.

In the light of all these facts it is not surprising that the dogfish egg should contain urea before the development of the embryo has begun, nor that the embryo should produce urea during its development. But before any closer interpretation of our data can be given it is necessary to consider the question of whether the oviparous dogfish egg (Scyllium canicula) forms a closed system or not, for if it should not, the urea formed by the embryo could easily escape into the water surrounding the egg, and the ascending curve might be a measure of the rate of escape as well as of the rate of formation.

At first sight there is every reason for supposing that the dogfish egg is not a closed system. In the earlier stages of development it is completely filled by the yolk and the white, but about half-way through development, slits appear at the four corners of the egg-case, which widen, lose their plugs of jelly and finally gape open, allowing sea-water to penetrate into the egg-interior. In the case of certain rays these slits are believed to assist the respiration of the embryo, and Clark(7), using suspensions of carmine granules, has shown that definite currents pass in and out of the eggs through them. And we often found bubbles inside the larger eggs after only slight handling.

We determined to test, therefore, to what extent the eggs of Scyllium retain urea and ammonia. First of all we made a few experiments with the egg-cases themselves, cutting discs from them about 1·5 or 2 cm. in diameter and fitting them up as diffusiometers, i.e. held with paraffin wax between two cork rings at one end of a glass tube. Urea solution was placed above the membrane and water without urea below, and the apparatus was left for some days, after which time the urea-content of the lower solution was estimated, and the diffusiometer tested with neutral red or some other dye to make sure that it had been properly sealed. The results were as follows :

Exp. 1. 12 c.c. containing 3·12 mg. urea/c.c., i.e. in toto 37·44 mg. urea, were placed in the upper part of the apparatus, with pure filtered sea-water below. After three days 1·5 mg. were found below the membrane.

Exp. 2. 62·4 mg. urea in sea-water placed above the membrane. After three days 1·3 mg. of urea were found below the membrane.

Exp. 3. 62·4 mg. urea in sea-water placed above the membrane but with the membrane reversed, so that the natural outside was on the upper side instead of on the lower. After three days 0·05 mg. urea was found below.

These facts, which other similar trials confirmed, seem to show that the horny egg-case membrane lets urea escape. The area through which active diffusion took place was 1 cm. in diameter, and if about 112 mg. of urea could pass through it in three days, a whole egg, having a surface of about 20 sq. cm. (Ford(16)) or 25·5 times the area of the piece of capsule taken, could lose about 38 mg. urea in three days, i.e. enough to drain the egg completely in a short time. The horny egg-case can therefore not now retain the urea of the egg-interior. More than one line of argument, therefore, seemed to indicate that the dogfish egg is not a closed system as regards urea.

We therefore determined to place Scyllium eggs at different stages of development in small vessels of sea-water and to estimate the urea-content of the surrounding liquid from time to time.

It may be said here that these determinations necessitated a slight change in the estimation-method. We found that when the phosphate buffer was added to seawater even if diluted, a precipitate was formed, probably of the calcium salt, and that this precipitate carried down with it or in some way inactivated the urease. If, however, the buffer was omitted, quite good results were obtained (94 per cent., 91 per cent, on known amounts). We had also to go into the question of the removal of urea from sea-water, in order to be sure that any urea lost by the eggs would remain intact for us to estimate. It is usually supposed that the many thousands of tons of urea which must be added annually to the sea are removed by diatoms and bacteria. Wishing to avoid the necessity of using a Berkefeld filter, we simply filtered the outside sea-water so that it contained no diatoms or organisms of similar size, and then tested the capacity of urea to remain unchanged in it ; 95 percent, was recoverable after ten days.

We then went on to compare the effect of the presence of Scyllium eggs.

Exp. 4. Four eggs were taken (in one the embryo was 3·5 cm., in another i-o cm. and in the remaining two 2·5 cm.) and placed in 500 c.c. fresh filtered outside sea-water. After three days the ammonia and urea were estimated. In the 500 c.c. there was 0·48 mg. of ammonia and 0·18 mg. of urea, or 0·09 mg. per cent, of ammonia, and 0·03 mg. of urea. In a similar lot of water which had not contained any eggs there was found 0·068 mg. per cent, of ammonia, and no urea. The urea lost by the eggs was therefore minimal in quantity, and probably not outside the experimental error. The eggs, moreover, contained within them between 30 and 50 mg. of urea.

Exp. 5. Five eggs were taken (embryos of 3·6, 3·8, 3·9, 3·0 and 2·5 cm.) and placed in 500 c.c. of sea-water as before. After four days the control showed 0·04 mg. per cent. ammonia1 and 0·04 mg. per cent, of urea, while the water surrounding the eggs showed 0-08 mg. per cent, of ammonia and 0·08 mg. per cent, of urea. This was especially remarkable as the five eggs between them contained well over 50·0 mg. of urea and the slits in the egg-cases were all well open, so much so that in three out of the five large bubbles were to be seen inside. We could only conclude that in spite of a free penetration of seawater into the egg-case, the system embryo-yolk was not giving up more than minimal amounts of its urea to the exterior.

If then there was no parallel between the horny egg-case of the dogfish and the strictly closed egg-shell of the chick, was it possible that the embryo and yolk formed a closed system or something approaching it? Experiments showed that this was so :

It is thus evident that 99 per cent, of the urea and about 92 per cent, of the ammonia of the dogfish egg is retained within the yolk or the embryo and does not appear in the white or jelly between them and the shell. Unfortunately we did not make any determinations on embryo and yolk separately, and we are therefore unable to say whether the urea formed by the embryo during its development is mainly retained within its body or is excreted into the yolk to be subsequently reabsorbed. An interesting consideration arises from the fact that if the main channel of elimination of the urea in the adult dogfish is through the bile and perhaps through the intestinal walls, the urea produced during development would tend to pass backwards into the yolk, for as Balfour(3) says, “the nutriment from the yolk-sac is brought to the embryo partly through the umbilical canal and so into the intestine, and partly by means of blood-vessels in the mesoblast of the sack.” If then the vitelline membrane and the blastoderm were impermeable to urea, as the gills are, the state of affairs experimentally found by us would naturally arise. The cloaca do not open in Scyllium until a comparatively late stage, and the oesophagus, which opens for a short time early on, closes again and becomes for a long time a solid cord of cells.

From the standpoint of general physiology the elasmobranch egg is of great interest. It has elsewhere been suggested that the nature of the end-products of protein metabolism arises from the requirements of the embryo (25). In aquatic eggs, such as the sea-urchin’s or the trout’s, the waste-nitrogen can be got rid of as ammonia or urea, dispersing readily in the surrounding water, but in the terrestrial egg, on the contrary, the non-diffusible insoluble uric acid has to be brought into play, as is seen in the eggs of sauropsida and insects. The word “cleidoic” has been introduced (26) to designate those eggs which are shut off thus from their surroundings as closed boxes (kλϵlδóω, shut up). It is evident that survival value attaches to the closed-box egg, for the longer an embryo can continue its prenatal existence the stronger it will be when it at length emerges, and for this end, much more protection is required than for the minute egg whose development is quickly accomplished. It is therefore very interesting that the only class of animals lower than the reptiles which have evolved a structure approaching the cleidoic egg, are just those which have found out a way to withstand a concentration of urea in their bodies which would ordinarily be rapidly fatal. If the Scyllium embryo had had to arrange for an efficient removal of its nitrogenous waste-products, it could perhaps not have elaborated its egg-case. But the evolution of the egg-case must have taken place in two stages; first the elasmobranchs, having discovered how to withstand severe uraemia, were able to enclose their embryos completely, and secondly when it later became convenient to open the box again, whether for the respiratory water current or other purposes, the selachians, having become adapted to urea-retention, continued to store it within themselves.

Our absolute values for urea are lower than those of all previous investigators, no doubt because we used the quite specific urease method instead of extractions with alcohol and the preparation of urea nitrate as was usually done before. The contents of a Scyllium egg in the early stages weighs about 5 gm. wet and 2 gm. dry so that as there is some 10 mg. of urea present then, the egg contains 200 mg. per cent. This is a great deal smaller amount than the figures given by Krukenberg (22). A Scyllium embryo after hatching, 11·15 cm-long, weighed 3·8 gm. and would contain, extrapolating from our curve, about 22 mg. of urea N, or 44mg. urea, i.e. 1160 mg. per cent.—more in accord with the older figures. We also made a few estimations on the eggs of the “spur-dog,” Acanthias vulgaris, which are much larger than those of Scyllium, with the following results :

There did not seem to be any great increase in the urea of the eggs, much more being provided by the maternal organism than in Scyllium. As the undeveloped egg weighs 25·2 gm. approximately, it will contain 888 mg. per cent, of urea, an interesting value because, as we have already seen, the blood-urea in closely-related forms has been found by more than one observer to be about 880 or 900 mg. per cent. The Acanthias egg then has exactly the same percentage of urea as the blood, unlike that of Scyllium where it is much lower. It is possible that the reason why the Acanthias egg does not show a steadily rising urea-content during its development is that being ovoviviparous, and held within the maternal uterus, it forms part of the maternal system, and having all the urea it requires provided at the start, depends much less than the Scyllium egg on the activity of the embryo itself.

But perhaps the most interesting result which emerges from the analyses of Acanthias eggs is that just as in Scyllium the major part of the urea is held in the embryo and yolk.

The partition in Acanthias, however, is not so striking as it is in Scyllium.

  1. A study has been made of the urea and ammonia production of selachian embryos, especially in Scyllium canicula.

  2. The observation of earlier workers, that the undeveloped dogfish egg contains notable amounts of urea, has been confirmed.

  3. During development, the urea-content of the egg increases considerably, the embryo adding to the original quota supplied by the maternal organism. In the egg of Scyllium the urea-content is thus increased four or five times.

  4. The egg-case of the Scyllium egg does not form a closed system to urea for its wall is permeable to this substance, and at any rate in the later stages there is a free penetration of sea-water into the egg-case through the four slits. Nevertheless, only minimal amounts of urea are found in water surrounding the developing eggs, and it is probable that the walls of the yolk-sac are impermeable to urea. For 98 per cent, of the urea of the egg is found in the embryo and yolk, so that an excretion must take place not into the white but into the yolk, as, indeed, might be expected if the main path of exit of the urea in the adult is through the intestinal tract.

  5. Evidence is adduced which makes it likely that a peak of protein catabolism exists in the embryonic life of the dogfish, just as in that of the chick and the frog, and probably of mammals.

  6. It is pointed out that the only fishes which have evolved an egg of the cleidoic type (though not now actually cleidoic) are just those which have evolved the power of withstanding severe uraemia. This is in accordance with the view that the main end-product of nitrogen metabolism is causally connected with the manner of life of the embryo of the form in question.

Our thanks are due to Mr Richard Elmhirst and the staff of the Millport Marine Biological Station for their cordial help and co-operation during our stay. We also wish to acknowledge assistance received from the Government Grant Committee of the Royal Society and from the Thruston Fund of Gonville and Caius College, and to thank the authorities responsible in each case. During the course of the work, one of us (D.M.N.) held a Beit Memorial Research Fellowship.

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This agrees well with Gad-Andresen’s (18) figure of 0·035 mg. Percent.