ABSTRACT
In the first paper of this series (16) a survey was made of the phosphorus distribution in developing invertebrate eggs with the object of sketching out the broad outlines of the metabolism of this element during ontogenesis. Representative echinoderms, Crustacea, and a podaxonian were examined with respect to the behaviour of total phosphorus, lipoid, phosphoprotein and nucleoprotein phosphorus, total, organic and inorganic water-soluble phosphorus, etc. But interesting as the results of such a survey were, there remained a number of phosphorus compounds which merited study, among which may be named hexosephosphate phosphorus, adenylic acid phosphorus, glycerophosphate phosphorus, phosphorus in combination with cycloses, and phosphagen phosphorus. It is with the latter of these that this paper will deal.
The earlier workers on muscle chemistry were accustomed to estimate what they called “inorganic” phosphorus in the muscle tissue of vertebrates. But in 1927 Eggleton and Eggleton (2) using frog muscle, showed that a very large part of this so-called inorganic phosphate was really organic phosphate of a very labile nature ; it is so unstable in acid solution that it is hydrolysed during the estimation in the usual methods, where, for example, the muscle is extracted with 4 per cent, trichloracetic acid and allowed to stand for some hours. This labile compound, which was found to undergo partial breakdown on fatigue and complete breakdown on rigor, was named by the Eggletons phosphagen. At about the same time, Fiske and Subbarow (5) working with cat muscle, isolated (as the crystalline calcium salt) from protein-free muscle filtrate, a substance which they identified as an equimolecular compound of creatine and phosphoric acid. This creatine phosphate accounted for all the labile organic phosphate of the Eggletons, who, shortly afterwards, confirmed the preparation of creatine phosphate(3).
Creatine phosphate was soon found to be present in much greater quantity in striped voluntary than in smooth involuntary muscle, and this led the Eggletons to examine a number of invertebrates with negative results (3). But Meyerhof and Lohmann (12) soon showed that in invertebrate muscle the place of creatine phosphate is taken by a precisely similar compound containing arginine instead of creatine, and which, having rather different conditions of stability, was not detected by the methods of the Eggletons. Creatine phosphate is unstable in acid solution, and, moreover, its breakdown is strongly catalysed by the molybdate necessary for colorimetric estimation of phosphorus. The stability of arginine phosphate, on the other hand, increases with increasing acidity, from a concentration of N/100 acid upwards, and its rate of breakdown is decreased thirtyfold by the presence of molybdate. Instead of estimating the phosphagen by observing the rate of breakdown to free phosphate in the presence of molybdate, and extrapolating backwards to zero time, as was done by the Eggletons for vertebrate muscle extracts, it is necessary with the invertebrate muscle extracts to observe the increase in free phosphate upon standing in N/20 trichloracetic acid for 15 hours at 28° C. Under these conditions the arginine phosphate is completely hydrolysed and the pyrophosphate of the muscle is left unattacked.
These differences in properties obviously allowed of simple methods for estimating one phosphagen in the presence of the other, and the problem of their comparative distribution therefore lay open at once. The Eggletons themselves were the first to make comparative observations (3) but they did not follow the matter up and after the discovery of arginine phosphate by Meyerhof and Lohmann (12), Meyerhof alone (11) examined a certain number of marine invertebrates. Finally the subject was thoroughly gone into by Needham, Needham, Baldwin and Yudkin(14) who extended the enquiry to most of the invertebrate phyla.
Cephalopod muscle was first studied in this connection by Meyerhof, who stated in his paper, without however giving any figures, that arginine phosphate was absent from the mantle musculature. But this absence was not confirmed by Needham, Needham, Baldwin and Yudkin(14), who observed a constant, though small, proportion of arginine phosphate in mantle, fin, funnel, and tentacle muscle, in Sepia and Octopus. They noticed, nevertheless, that if the animals were not in good condition (floating at the surface of the water, giving little resistance to capture, etc.) the muscle would then contain no phosphagen, and they suggested that as the animals in Meyerhofs tables were sometimes characterised by the phrase “lang in aquarium,” it was probable that the presence of arginine phosphate could only be demonstrated if the cephalopod was in perfect health and freshly caught.
During the course of a period of work at the Roscoff marine biological station, we found at our disposal a considerable quantity of the eggs of Sepia officinalis, and we therefore devoted some time to the estimation of the phosphagen in the embryos at different stages of development.
The method used was that of direct separation under ice-cold conditions. Before being dissected and weighed the material was well cooled in ice. The weighed portion was then introduced into ice-cold 5 or 10 per cent, trichloracetic acid (in the proportion of io c.c. per gm.) and thoroughly ground up with ice-cold quartz sand. After waiting io min. to allow of complete extraction, the mixture was filtered through a cooled funnel and the filtrate neutralised to phenol-phthalein with ice-cold phosphorus-free saturated soda. 4 c.c. of the neutralised filtrate was then pipetted out into a small cooled centrifuge-tube and treated with 1 c.c. of a 10 per cent, calcium chloride solution saturated with calcium hydroxide. After allowing the tube to stand for 10 min. in ice, it was centrifuged, the centrifugate poured into a 50 c.c. graduated flask and the precipitate washed once in the centrifuge with 4 c.c. of ice-cold distilled water and 1 c.c. of the calcium chloride solution. The precipitate, being dissolved in a drop or two of normal sulphuric acid, gave the true preformed inorganic phosphorus, and the filtrate, after having stood at room temperature for 15 min. in the presence of the molybdate reagent, gave (or would have given, had there been any) the creatine phosphate phosphorus. Next, another portion of the original trichloracetic acid filtrate (before neutralisation) was taken, and sufficient distilled water added to make the concentration of trichloracetic acid about 0·1 N (usually five volumes). This was then allowed to remain overnight (15 hours) in an electrically-regulated incubator at 28° C., after which the estimation of its contents gave the inorganic plus creatine plus arginine phosphorus, and hence by subtraction, the latter alone. In the subsequent summary of data (Table II) the inorganic phosphorus estimation will be referred to as fraction A, the creatine as fraction B, the total as fraction C, and the arginine, C −(A + B), as fraction D. Our estimation method throughout was the colorimetric technique of Fiske and Subbarow (4), a variant of the original Briggs method, which consists in converting the inorganic phosphate quantitatively into phosphomolybdic acid and measuring the intensity of the blue colour formed when this is reduced with 1-, 2-, 4-amino-naphtholsulphonic acid. The molybdate solution used was one of 2·4 per cent, ammonium molybdate in 5N sulphuric acid, and the reducing agent was dissolved in sodium sulphite and bisulphite solution according to the method of Luck (9).
Before during the last part of, a few words may be said about some other quantitative data which we were able to collect in the course of our investigation. Table I shows the details of our material, consisting of some 1100 embryos and yolks. We were not able to observe the length of time taken from laying to hatching and we cannot therefore allot a time scale on the basis of our own notes. A body-weight/time relation for Sepia is, however, given by Ranzi (19), and the column in Table I showing day of development is calculated from this source. Table I also contains a statement of the stage reached by the embryos according to the recent “Normaltafeln “of Naef (13) for Sepia. An interesting relation is found by plotting the weight of the yolk against the weight of the embryo (Fig. 1). At first the yolk weight sharply descends, but the rate of decrease steadily falls off, and by the time that the embryo has reached a weight of about 100 mg. (half the weight at which, in our experiments, it hatches), the decrease has almost entirely ceased. This is no doubt to be explained by the absorption of water and salts from the saline medium, which has been shown by Ranzi (18) to take place during the development of Sepia. The water content of the whole egg, he finds, rises continually (in absolute values), slowly from the beginning of development until the 55th day, more rapidly from that time onwards. This process will to some extent compensate for the loss of weight from the yolk due to passage of solid and water into the embryo, and as can be seen from Fig. 1 this compensation reaches a very effective level during the last part of development.
The chemical data are summarised in Table II. It may first of all be remarked that fraction B never contains any phosphorus, indicating the absence of creatine phosphate. This was always very definite in the embryonic material, but in the analyses of adult Sepia muscle, some of which are given at the bottom of the table, traces of phosphorus did sometimes appear in this fraction. In another communication, however (14), we have given good reasons for regarding these traces as due to the high concentration of inorganic phosphate present in cephalopod muscle, and the consequent difficulty of obtaining complete separation.
In the yolk no phosphagen was ever found. But in the embryos phosphagen was always present (fraction D, arginine phosphate) in amounts varying from 0 · 011 to 0·106 mg. phosphagen P per gm. wet weight of embryo. In the earliest and latest stages these levels were lower than in the middle stages, with an average at 0·060 mg. per gm. wet weight. But when compared with the amounts of inorganic phosphate present in each case, a more unmistakable regularity appeared, the phosphagen present in per cent, of the total phosphorus rising to a maximum and thereafter falling. This is plotted in Fig. 2. The maximum appears to occur at an embryo weight of 95 mg., i.e. at about 86 days of development, or rather towards the conclusion of the period before hatching (see Fig. 3).
Before discussing the significance of this peak, we may draw attention to the results obtained in the inorganic phosphate fractions. As Fig. 4 shows, the in-organic phosphate per gm. wet weight of embryo rises rather suddenly between the embryo weights of 10 and 40 mg. (40–65 days) and then, adopting a slighter slope, continues rising for the rest of development. In the yolk there is practically no inorganic phosphate to begin with, but this rises about the time when that in the embryo is rapidly rising, and reaches a maximum of 0·05 mg. per gm. wet weight at embryo weight of 25 mg. (55 days), after which it falls to a constant level of 0·02 gm. per gm. wet weight. These relationships are in some contrast with those which have been so fully worked out for the developing hen’s egg by Plimmer and Scott (17) and Masai and Fukutomi(10). In the yolk of the hen’s egg, the in-organic phosphate remains throughout development at an infinitesimal level, phosphorus from the lipoids being liberated in water-soluble organic form, and so transported to the seats of calcification in the embryonic bones. In the embryonic body the inorganic phosphate rises parallel with calcification. In the Sepia egg, on the other hand, we must assume a very active liberation of lipoid phosphorus at the beginning of development, followed by some failure on the part of the embryo to absorb completely the inorganic phosphate available, and eventually the attainment of a steady level of equilibrium. It must be remembered, however, that of the 3·3 mg. of ash with which the embryo Sepia hatches, no less than 2·5 mg. are obtained, according to Ranzi(19), from the sea, and in this quota some phosphate must no doubt be contained. It is at any rate clear that the general scheme of phosphorus metabolism in the cephalopod egg differs considerably from those already known for other eggs, and further investigation of it would be very desirable.
As regards the peaked curve of Fig. 3, its significance seems to us to lie in the varying growth-rates and maximal weights of the different organs and parts of the developing body. We regard it as a chemical index of the proportion taken in the body as a whole by the muscle systems. Although, unfortunately, there are no data extant which show us the growth of the parts in cephalopod embryos, there are for other animals a number of such investigations. Schmalhausen (21), for instance, working on the chick embryo, has given a graph showing the relative weight of its various parts in per cent, of the total body weight on each day of development. It is here very noticeable that whereas the brain, the heart, the lens, and the mesonephros have peaks between the 2nd and 6th days, the fore-limb (which may be regarded primarily as a muscle-mass) has its peak very late in development, on the 17th day. Precisely similar results come from the work of Jackson (6) on man, where it is seen that the muscle masses of the fore and hind extremities attain their maximum development (in per cent, of the whole body weight) about the 9th month; the head, for example, having attained and passed its maximum during the 2nd month. In the dogfish, as studied by Kearney (7), there is no maximum for the muscles before hatching, at which time they form 45·0 per cent, of the total body weight, but towards the adult stage they increase still further and finally form 63·0 per cent, of its weight. Jackson’s conclusions for the human embryo were confirmed and extended by Scammon (20) who analysed a large number of observations collected by Corrado (1), and showed that the constant b in the well-known Calkins equation was always negative for the extremities, since they grow more slowly than the body as a whole. Similar results are seen in the work of Keene and Hewer (8). The position may be summed up in the words, “The muscle masses, shown especially in the figures for fore and hind limbs, increase steadily in relative weight, and reach their maximal relative size at or shortly before birth” (15) (p. 452).
How far this generalisation can be applied to the cephalopod is, of course, uncertain, but a glance at the “Normaltafeln” of Naef (13) is sufficient to show that at a stage (e.g. stage 13) when about one-third of the incubation-time has elapsed, the mantle is so small as hardly to equal the cephalic end, arms, eyes, etc., in size, although in the adult at least 60 per cent, of the distance between the tip of the body and the tip of the tentacles is taken up by the mantle, and the viscera beneath the mantle cavity.
SUMMARY
In the course of an investigation on the phosphorus metabolism of the developing egg of Sepia, it was found that the embryo, but not the yolk, contains arginine phosphate at all stages.
The inorganic phosphate per unit weight of embryonic tissue rises continually throughout development, but the inorganic phosphate per unit weight of yolk rises to a maximum early in development and falls thereafter to a constant level.
The arginine phosphate of the embryo, expressed as per cent, of the total inorganic and labile water-soluble phosphorus, rises to a maximum at embryo weight 95 mg. (86 days’ development) and then falls to the adult level by the time of hatching. It is suggested that this reflects in chemical terms the attainment of maximal relative weight of the muscle masses during development.
Acknowledgements
The authors wish to acknowledge with thanks financial assistance from the following sources: The Thruston Fund of Gonville and Caius College (J. N.); Christ’s College (J. Y.); the United Services Fund of the British Legion (E.B.); the Government Grant Committee of the Royal Society. They also wish to thank Prof. Ch. Perez and the staff of the Marine Biological Laboratory at Roscoff for their kind welcome and constant help.