THE Joumar, cm Bro~oorca~ CHEMISTRY Vol. 235, No. 10, October 1960

Printed in U.S.A.

Biochemistry of Anuran Metamorphosis

VII. CHANGES IN SERUM PROTEINS DURING SPONTANEOUS AND INDUCED METAMORPHOSIS*

ALBERT E. HERNERt AND EARL FRIEDEN

From the Department of Chemistry, Florida State University, Tallahassee, Florida

(Received for publication, April 1, 1960)

In the evolution of the vertebrates, amphibia bridge the gap be- tween the fresh water fishes and the land dwelling reptiles. Further, most of the Anura undergo metamorphosis, which es- sentially consists of the transformation of a fresh water form, the tadpole, into a terrestrial form, the frog or toad. Accordingly, the study of the frog and its larval forms provides an excellent vantage point for the study of comparative biochemistry.

Biochemical measurements of the changes accompanying ver- tebrate metamorphosis have been briefly reviewed by Wald (2). A review dealing specifically with amphibian metamorphosis will appear in the literature.1

Previous studies of protein changes during amphibian meta- morphosis have dealt with variations in enzymic activities (cf. (3, 4, 5)) and changes in the hemoglobins (6, 7). In the light of these changes and the general cellular differentiation which ac- companies metamorphosis, it is of interest to examine the serum proteins for ramifications of the changing requirements of the differentiating cells. In addition, the change toward a morpho- logically more complex animal, living in a different environment, would conceivably also be contributory to a changing serum protein distribution.

A difference between the serum protein complement of tadpoles and frogs is suggested by the observation of Braus (8) that anti- sera against adult tissue extract of Bombina vuriegata failed to react with tissue extract of the tadpoles of the same species. However, in a similar study, Wilkoewitz and Ziegenspeck (9) were unable to distinguish between the adult and larval stages of Rana esculenta.

Preliminary reports from this laboratory on changes in the electrophoretic patterns of serum proteins during anuran meta- morphosis have appeared (10, 11). This paper presents the changes in the nature and content of serum proteins for a wide spectrum of stages and species of anurans.

EXPERIMENTAL PROCEDURE

Methods and Materials

Animals-Rana grylio (southern bullfrog) and Rana heckscheri (swamp frog) tadpoles and frogs were collected locally. Rana

* Supported in part by Grant No. C-3006 from the United States Public Health Service. This work represents a portion of the dissertation submitted by Albert E. Herner to the Graduate School in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

The previous paper in this series is concerned with changes in nucleic acid metabolism (1). More general information on the biological events involved appears in References 2, 15, 19, 21, 22, and 31.

t Present address, Facnltt! des Sciences, Universitc libre de Bruxelles, Bruxelles, Belgique.

1 T. P. Bennett and E. Frieden, in preparation.

catesbeiana (North American bullfrog) tadpoles and frogs were purchased from the Lemberger Company, Oshkosh, Wisconsin, and the Carolina Biological Supply Company, Elon College, North Carolina. Rana clamitans (green frog) frogs wereobtained from the Florida area, and Xenopus Zaetis (smooth clawed frog) frogs from Jay E. Cooke, Cockeysville, Maryland. Xenopus Zaewis tadpoles were hatched in the laboratory from fertilized eggs produced by treatment of the adult frogs with a chorionic gonadotrophin preparation obtained from Armour and Company, Chicago, Illinois.

Tadpoles subject to induced metamorphosis by treatment with Ts2 were injected intraperitoneally once with 10 ~1 per g of body weight of a low3 M solution using a syringe fitted with a No. 27 needle.

Studies were conducted upon groups of animals arranged ac- cording to the stage of metamorphic development. Animals des- ignated in Table I to be in early metamorphosis were tadpoles with very little hind leg development, reflected by a low L/T ratio. Intermediate animals were those with both appreciable hind leg development and remaining tail. Postmetamorphic frogs were animals which had just completed spontaneous metamor- phosis, the last vestige of tail being resorbed and the animals having the appearance of diminutive though completely formed frogs. The term adult frog refers to the reproductively mature animal. Weights are indicated for the Xenopus frogs since the 3-g frog is larger than the postmetamorphic animal and the 20-g animal is smaller than the full grown adult.

The T,-treated animals were treated by injection as previously described and maintained for the number of days indicated in Table I without further treatment before bleeding. Appropriate control experiments were performed for R. heckscheri, the tad- poles being treated by injection with a 0.70 g/100 ml solution of saline instead of T3. Results obtained for these animals were in all respects similar to the untreated tadpoles at early meta- morphosis.

Collection of Blood and Sera-Tadpoles were first immobilized by immersing in a 1: 3000 solution of tricaine (Sandoz Pharma- ceuticals, Hanover, New Jersey) for 5 minutes. Frogs were anesthetized with ether. The hearts of the animals were exposed and the pericardial cavity dried with tissue paper to minimize contamination of samples by body fluids. A collecting tube, made from 5-mm soft glass tubing extended to a capillary, then was inserted by means of the capillary tip into the conus arteri- osis. Heart action pumped blood up the tube. It was found that yields could be appreciably increased if, after a minute, the tube

2 The abbreviations used are: L/T ratio, ratio of hind leg length to tail length; A/G ratio, ratio of per cent albumin to per cent globulin; T3, 3,5,3’-triiodo-L-thyronine.

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Chang es iv hind-leg to tail and albumin to globulin ratios during anuran metamorphosis

Designation No. of animals Description I

L/T Ration

-

.- Rana grylio

Tadpole-l Tadpole-2 Tadpole-3 Tadpole-4 Tadpole-5 Frog-l Frog-2

Rana catesbeiana Tadpole-6 Tadpole-7 Frog-3 Frog-4

Rana heclcscheri Tadpole-S Tadpole-9 Tadpole-10 Tadpole-11 Tadpole-12 Tadpole-13 Tadpole-14 Frog-5

Rana clamitans Frog-6

Xenopus laevis Tadpole-15 Frog-7 Frog-S

9 Early metamorphosis 3 3 days after T8 injection 6 5 days after Ta injection 4 6 days after Ta injection 3 Intermediate 7 Postmetamorphosis 3 Adult

0.09 f 0.04 0.18 ziz 0.03 0.28 f 0.08 0.36 f 0.07 1.22 f 0.12

5 Early metamorphosis 7 6 to 7 days after T) injeation 4 Postmetamorphosis 4 Adult

0.04 f 0.03 0.39 AZ 0.09

10 Early metamorphosis 3 1 day after Ts injection 5 2 days after Ta injection 7 3 days after Ta injection 3 5 days after Ta injection 6 6 days after Ta injection 2 Intermediate 5 Adult

0.11 f 0.02 0.07 f 0.02 0.10 f 0.05 0.14 f 0.05 0.18 f 0.03 0.41 f 0.16

0.60

2 Adult

12b Early metamorphosis 0.20 2 Weight ea. 3 g. 0.52 2 Weight cu. 20 g. 1.49

Serum Proteins during Anuran Metamorphosis

TABLE I

Vol. 235, No. 10

0 Including standard deviations! v = Zziz - QZi)z/n

n-l * Two groups of 6 animals each.

was removed, most of the capillary broken off, and the tube re- inserted into the original puncture.

To obtain samples from the Xenapus Zaetis tadpoles the dermal layers covering the pericardial cavities were removed and the in- tact cavities were punctured with melting point tubes extended to fine capillaries. After rupture of the blood vessels within the cavity, blood, probably partially mixed with body fluids, col- lected in the tube by virtue of heart action and capillarity.

Blood samples for all of the animals were collected for a period of 20 to 30 minutes. This time interval was sufficient both for maximal yield and to allow for complete clotting within the col- lecting tubes. Clots were separated from the sera by centrifu- gation at room temperature.

Paper Electrophoresis-The method used was that of Jencks, Durrum and Jetton (12) as modified by Downs et ~2. (13), using the Spinco paper electrophoresis apparatus (Spinco Division, Beckman Instruments, Inc., Palo Alto, California) with barbital buffer containing Sterox SE (Aloe Scientific, St. Louis, Missouri), 0.2 ml/100 ml of buffer.

The volumes of serum used for each group generally were de- pendent upon the relative protein concentrations and are given in the legends of Figs. 1 to 4. The A/G ratio of R. catesbeiana frog serum was found to be unaffected by differences of as much as 4-fold in volume.3

3 A. E. Herner, Ph.D. dissertation, Florida State University, 1960.

A/G Ratioa

0.12 i 0.03 0.16 i 0.04 0.22 f 0.05 0.35 I 0.08 0.54 f 0.14 0.81 f 0.11 0.90 f 0.01

0.12 f 0.02 0.23 3~ 0.08 0.64 & 0.09 0.70 f 0,07

0.02 f O.o’L 0

0.08 zk 0.05 0.07 f 0.02 0.09 f 0.02 0.11 f 0.03

0.14 0.48 zt 0 05

0.31

Total Serum Protein Concentration-Total serum protein was determined as the trichloroacetic acid precipitable nitrogen (X 6.25) and by the biuret method of Gornall et al. (14) modified to a semimicro scale. The latter procedure was simpler and gave better precision. Total protein values for the Xenopus animals were estimated from the area under the electrophoretic patterns.

Determination of Albumin to Globulin Ratio-In work with human sera, Downs et aZ. (13) have shown that the values for the relative percentage of albumin, from the electrophoretic patterns using the methods discussed previously, are comparable to al- bumin levels found from salt precipitation methods. Accord- ingly, the values for the A/G ratios given in Table I are derived from the electrophoretic patterns and are averages of the ratios of the area under the fastest moving fraction to those under all the remaining fractions.

Ekctrophoretic Patterns-The original electrophoretic patterns are densitometric tracings prepared from the bromophenol blue stained paper strips (see Fig. 5) upon which the electro- phoretically separated serum proteins are adsorbed. Figs. I to 4 are composite drawings derived from the original electropho- retie patterns. The number of animals from which sera were.ob- tained to prepare the electrophoretic patterns is indicated in Table I as is a brief description of the designated groups to which the animals belong. Each serum sample was run in du- plicate when sufficient serum was available. In preparing the composite patterns, points were selected at the maxima, minima,

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October 1960 A. E. Hewer and E. Frieden 2847

and plateaus of the original electrophoretic patterns, the dupli- cate values averaged for each sample and then a mean and the standard deviation determined for each point for the several animals in the group. A smooth curve is drawn through these mean values. The standard deviation for each point is repre- sented by the vertical bars drawn through the points. Where standard deviations are not indicated there are but two animals in the respective groups.

The patterns for the Xenopus Levis tadpole (tadpole-15, Fig. 4) represent pooled samples from two groups of six animals each. This number of animals was required to obtain a sufficient sam- ple (100 ~1) to determine the protein distribution. A human serum pattern is included in Fig. 4 for the purpose of compari- son.

REST

Increase in A/G Ratio and Albumin Concentration with Metamorphosis

The most striking change in the four species studied is the increase in, and the eventual predominance of, the fastest mov- ing fraction. corresponding to the increasing metamorphic de- velopment of the tadpoles. This fraction has the same electro- phoretic mobility as human serum albumin in the case of the Ranidae and also shares the intensive staining properties of human serum albumin. Under our experimental conditions the fraction has an anodal migration of 7 cm for the Ranidae and 8 cm for the Xenopus samples.

The migration of this fraction remains relatively constant for all stages of a given species so that its evolution may be con-

Rana heckscheri --

- 14

-5

-I 0 I 2 3 4 5 6 7 8 9

MIGRATION IN CM

FIG. 1. Composite electrophoretic patterns for the serum pro- teins of Rana heckscheri at various stages of metamorphosis. Spontaneous metamorphosis: tadpole-$ early metamorphosis; tadpole-14, intermediate; and frog-5, adult. Tp-induced meta- morphosis: tadpole-11 and -13, 3 and 6 days after Ta injection. Details for the group designations are in the text and Table I. Globulin Fractions a, b, c, and d are indicated. The fraction with the greatest migration is the albumin. Serum volumes: tadpoles, 40 fit1 and frog-5,20 ~1.

Rana grylio

TADPOLE - 5

FROG- I

FROG - 2

I I I I : : : : : : I -101 23456789

MIGRATION IN CM.

FIG. 2. Composite electrophoretic patterns for the serum pro- teins of Rana grylio at various stages of development. Spon- taneous metamorphosis: tadpole-l, early metamorphosis; tad- pole-5, intermediate; frog-l, postmetamorphosis; and frog-a, adult. TI-induced metamorphosis: tadpoles-2, 3, and 4, respec- tively, 3, 5, and 6 days after Ta injection. The volume of serum applied was 20 ~1 in all cases.

veniently surveyed as the tadpole develops into the adult frog. Further, it has the same migration for all stages of the Ranidae species studied. The actual chemical relationship of the fast moving fractions to each other or to human serum albumin is open to conjecture; however, based on identical electrophoretic mobilities, we may postulate that they are similar. Along these lines, this fraction in the Xenopus animals would appear to be less similar to human serum albumin. However, as Anfmsen (15) points out, these differences may not be of the kind which involve the functionally critical parts of the serum albumin structure. In this paper, we shall refer to the fastest moving fractions as albumins and the remaining fractions as globulins.

Changes in A/G Ratio and Albumin Concentration in R. hecks- cheri-Changes in the R. heckscheri patterns (Fig. 1) are most dramatic in that the young tadpole (tadpole-@ starts with an extremely low level of albumin (7 X 10e3 g/100 ml)* which in- creases to a level of 0.7 g/100 ml in the mature adult (frog-5). This is reflected in an increase in the A/G ratio from 0.02 to 0.48 (Table I). This effect is deemphasized in Fig. 1 since only half the volume of serum was applied to the paper strips for frog-5 (20 ~1) as for the R. heckscheri tadpoles (40 ~1). The in-

* This extremely low level of albumin in tadpole-8 led us to speculate that a portion of the albumin might be trapped by asso- ciation with a globulin, e.g. Fraction d. However, the addition of varying amounts of human albumin to the sera of tadpole-8 showed, upon electrophoresis, that this albumin moved to the appropriate albumin position and independently of the globulins.

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2848 serum Proteins during Anuran Metamorphosis Vol. 235, No. 10

termediate animal (tadpole-14) falls into place with respect to the height of the albumin peak and a value of the A/G ratio of 0.14. The increase in albumin coincides with the progress of spontaneous metamorphosis.

Rana catesbeiana

TADPOLE - 7

FROG - 3

---_-_ - ___________________

FROG - 4

-I 0 I 2 3 4 5 6 7 8

MIGRATION IN CM.

FIG. 3. Composite electrophoretic patterns for the serum pro- teins of Rana catesbeiana at various stages of development. Tad- pole-6, early metamorphosis, tadpole-7, 6 to 7 days after Ta injection; frog-3 postmetamorphosis; and frog-7, adult. Twenty ~1 of serum were applied for the tadpoles and 10 ~1 for the frogs.

Xenapus laevis --

-- _--_____ -___ A TADPOLE - I5 ------- -

X. laevis -- n

Rana clamitans

X. laevis rfioc-8

I I : I I : : ; I ; I I -I 0 I 2 3 4 5 6 7 8 9 IO

MIGRATION IN CM.

FIG. 4. Composite electrophoretic patterns for the serum pro- teins of Xenopus Zaevis at three stages of normal development, and for the adult Rana clamitans. The albumin peak for the X. Zaevis animals is displaced to the right of the position for this peak in the Ranidae and adult human sera which is shown. Two peaks are present in the albumin area in the serum of the adult R. clamitans. Serum volumes: tadpole-15, 100 ~1; frogs-g, 7, and 8, 20 ~1; and adult human, 10 ~1.

For metamorphosis induced by TO there is no noticeable A/G effect after an incubation period of 1 day (tadpole-g, Table I) but at 2 days (tadpole-lo) there may be a statistically significant increase (Fischer t test; P of less than 0.10) in the A/G ratio from that of the untreated young animal (tadpole-8). This precedes any significant morphological effect, There is no further increase in the A/G ratio for an incubation period of an additional day (tadpole-11), but after this there is a gradual increase so that the values for tadpole-11 and tadpole-13 appear to be statistically different (P = 0.1).

It appears that in R. heckscheri the morphological response to TB is gradual. During the first 5 days of incubation, the L/T ratio increases by a factor of two. The L/T ratio increases quite rapidly for the next 2 days, more than doubling again. On the other hand, response on the part of the A/G ratio is maximal during the second 24 hours of incubation, increasing only slowly thereafter.

There is evidence of a fraction in the R. heckscheri frog serum with almost as great a mobility as the mayor albumin fraction. This can be seen as a shoulder on the globulin side of the albu- min peak in Fig. 1 (frog-5). It can be seen more clearly by reference to Fig. 5, which is a photograph of typical paper strips for frog-5 and the other R. heckscheri groups. This fraction and the intensely stained albumin fraction are indicated by the arrows on the frog-5 strip. The former fraction is not present in the other R. heckschm-i animals.

Changes in A/G Ratio and Albumin Concentration in R. grylio and R. catesbeiana-R. grylio (tadpole-l) and R. catesbeiana (tadpole-6) tadpoles have a higher A/G ratio (0.12 for both) and higher albumin concentrations (R. grylio = 0.15 g/100 ml, R. catesbeiana = 0.12 g/100 ml) than the comparable R. hecks- cheri tadpole (tadpole-g). The A/G ratios in the mature ani- mals of both of these species (frog-2 and frog-4) are also at higher levels than the corresponding values for the frog of R. heckscheri. The albumin concentrations for the frogs of R. grylio and R. catesbeiana are similar (about 1 g/100 ml) and higher than that for the R. heckscheri frog (see above).

The response of R. grylio to Ts treatment was tested as a func- tion of time. Reference to Table I indicates that both the

morphological response and the rate of increase of the A/G ratio were approximately linear, differing from R. heckscheri discussed above. However, the L/T ratio increased by a factor of four for both species over the total 5- or 7-day incubation period. Similarly, the A/G ratio of R. grylio increased by a factor of three and that of R. heclcscheri by a factor of five, al- though the R. heckscheri ratios (tadpole-8) are subject to a par- ticularly large experimental variation.

The morphological response of R. catesbeiunu tadpoles to TB treatment was similar to that of R. grylio and R. heckscheri though the A/G ratio of the former increased by a factor of only two.

Included in Figs. 2 and 3 and Tables I and II are results ob- tained for the species R. grylio and R. catesbeiunu both for post- metamorphic frogs (frog-l and frog-3) and for the mature ani- mals (frog-2 and frog-4). The data for these species show that after the animals complete metamorphosis their serum protein picture remains essentially unchanged. Growth of the animals does not lead to any further change either in the distribution of these proteins or the total concentrations.

Two distinct, lightly stained bands were invariably found in the albumin region for tadpoles-l and -2 of R. grylio. Just one

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October 1960 A. E. Hewer and E. Frieden 2849

with a much higher albumin peak, also has a larger total serum protein concentration of 2.56 g/100 ml. Boyden and Noble (17) have reported a similar value of 2.66 g/100 ml for the total serum protein concentration of the R. catesbeiana frog.

Changes Among Globulins of Ranidae-Because of the multi- plicity and interactions of the globulins (18), the changes which take place in these proteins during metamorphosis must be in- terpreted cautiously. A change in the amount of a given frac- tion may be the result of either a change in chemical structure which will affect its mobility or its tendency to interact with other materials, or to an actual increase or decrease in the rate of biosynthesis of this fraction. Subject to these reservations the globulin fractions have been arbitrarily labeled, in order of increasing mobility, from a to d in Figs. 1, 2, and 3. It is not to be assumed that Fractions a to d necessarily represent identi- cal proteins among the different species and groups of animals. However, their designation, based upon electrophoretic mobility, does provide a means for developmental comparisons as well as interspecies relationships of the globulins.

FROG -8

During the spontaneous metamorphosis of the three principal Rana species, there is a shift of the globulins from Fraction c to Fraction a and, to a lesser extent, Fraction d. Only in R. heck- scheri is there an appreciable increase in Fraction b. The Ta treated animals reflected these increases in Fractions a and b but did not show an appreciable shift in Fraction d.

Globulin Changes for X. laevis-There is an increase in all of the globulins during metamorphosis and the appearance of faster moving globulins in the more advanced animals (Fig. 4). These latter fractions conceivably are present in the tadpole sera, al- though not in measurable amounts. During growth of the frogs, the globulins appear to redistribute, the most noticeable effect

FIG. 5. Top $ve strips, Rana heckscheri. The albumin peak in being the diminution of the fractions with a mobility in the mid-

tadpole-8 is very small, increasing during metamorphosis to a dle range. The globulin distribution of the larger Xenopus frog

maximum in frog-5. In this latter group, there is a less densely staining fraction moving almost as fast and merging with the TABLE II deeply stained main albumin fraction (see arrows).

Bottom three strips, Xenopus laevis. The changes in the serum Variations in total serum protein concentration during

proteins of the fully metamorphosed animal upon maturation can anuran metamorphosis

be seen by a comparison of the strip for the small frog (frog-7) with that for the larger frog (frog-S). Species

! Group No. of

animals rotein concentratid

band was evident in the albumin region of the more advanced R. grylio and in all of the R. catesbeiana. Rana grylio

Changes in A/G Ratio of X. laevis-The electrophoretic pat- terns of the X. laevis animals are presented in Fig. 4 and the A/G data in Table I. Typical bromophenol blue stained paper strips are shown in Fig. 5. The A/G ratio for the tadpoles of this species (tadpole-15) is considerable (0.20). It increases to Rana c&sb&na 0.52 with the completion of metamorphosis. At variance with the Ranidae species discussed above, the A/G ratio and the total protein concentration continues to increase as the frog matures. The A/G ratio reaches 1.49 and the total protein Rana heckscheri concentration becomes 3.5 g/lCO ml for the 20-g frog (frog-S).

Albumin in R. clamitans Frog-Presented in Fig. 4 is a corn- posite electrophoretic pattern for the sera of R. clamitans frog.

Xenopus laevis

It is similar to a pattern obtained by Dessauer and Fox (16) for this animal by a comparable method. This pattern is interest-

Tadpole-l 7 Tadpole-2 3 Tadpole-3 4 Tadpole-4 4 Frog-l 4 Frog-2 4 Tadpole-6 7 Tadpole-7 8 Frog-3 3 Frog-4 4 Tadpole-8 7 Tadpole-13 8 Frog-5 4 Tadpole-15 12c Frog-7 2 Frog-8 2

grams/100 ml

1.42 f 0.30 1.28 f 0.27 1.47 f 0.10 1.41 f 0.40 2.19 f 0.17 1.97 i 0.12 1.16 f 0.19 1.74 f 0.395 2.56 f 0.50b 2.56 f 0.53b 0.35 f 0.04 0.92 f 0.22b 2.03 f 0.19

0.2d 2.3d 3.5d

ing because of the relatively low albumin fraction and the ap- 2W - pearance of two distinct peaks in the albumin area. The low a Including standard deviations: (r =

n-l relative proportion of albumin is associated with a correspond- ingly low total serum protein concentration of 1.58 g/100 ml reported by Boyden and Noble (17). The adult R. catesbeiana,

6 Trichloroacetic acid insoluble nitrogen X 6.25. c Two pooled samples of serum each from 6 animals. d Estimated from the electrophoretic patterns.

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2850 Serum Proteins during Anuran Metamorphosis Vol. 235, No. 10

(frog-$ is more comparable to that of adult human strum than to the globulin patterns of the Ranidae frogs. In the latter the faster moving globulins (Fraction d) predominate, whereas the Xenopus frog and adult human sera the slower moving globulins are most prominent.

Comparison of Shapes of Electrophoretic Patterns of Ranidae- The patterns of the R. catesbeiana tadpole in early metamorpho- sis (tadpole-5) and both the post metamorphic and adult frogs (frogs-a, -4) of this species have the same general shape as the patterns of comparable animals of the species R. grylio (tadpole-l and frogs-l, -2). The difference for these two species in their terminal Ta-treated anima,ls (tadpole-4 and tadpole-7) is noted above.

Although the albumin level is low in the terminal T&reated animal (tadpole-13) and the intermediate animal (tadpole-14) of R. heckscheri, the globulin distribution of these animals is comparable to that in corresponding animals of R. grylio. The frog patterns of all three species are almost identical, with the slight variation mentioned previously for R. heckscheri frogs. It is surprising that the adult frog of another Ranidae species, R. clamitans (Fig. 4) has so low an albumin fraction in compari- son to the other three Ranidae studied. However, even here, the globulin distribution is similar to these other species.

The young tadpole serum patterns of R. heckscheri are dis- similar to those of R. grylio and R. catesbeiana. Yet these three species change to a similar pattern at the completion of metamor- phosis. This effect has its counterpart in the morphology of em- bryological development. de Beer (19) notes that there are cases of structurally divergent embryos which will, in the course of their development, become similar in structure. This phe- nomenon was given the name caenogenesis by Haeckel (20).

Total Protein Concentration-Table II shows that as an animal undergoes metamorphosis, either Ts-induced or spontaneous, there is usually an increase in the total protein concentration associated, to a large extent, with an increase in the albumin concentration. Of the Ranidae, on the basis of percentage in- crease, the effect is most marked for R. heckscheri and least so for R. grylio. The Ts-treated R. grylio does not show a signifi- cant change in serum protein. For the Xenopus frogs there is a significant increase in the total protein concentration with growth.

DISCUSSION

It has been known for some time (21, 22) that the highest se- rum protein concentrations are associated with the most highly evolved animals. The question as to how this increase is dis- tributed among the electrophoreticnlly separable components is not simply answered by phylogenctic studies (16, 23-26), because of individualities peculiar to the various species, which make a correlation study difficult. A study of the Anura, at various stages of metamorphosis, includes fishlike animals, as well as essentially terrestrial ones and animals at intermediate stages, all within each of the several species.

Anuran metamorphosis may be compared to the embryonic development of higher vertebrates. Moore, Shen, and Alexander (27) have followed the changes in the serum proteins during the embryological development of the higher vertebrates and found that the sera of developing chick and pig embryos increase in to- tal protein concentration. The serum albumin of chicks and pigs becomes the predominant fraction during the later developmen- tal stages. In the early embryos, the QI- and &globulins pre-

dominate. The component with a mobility similar to mam- malian y-globulin does not appear until rather late.

The increase in albumin in the Ranidae can bc ascribed to new needs which the animal requires in developing from the water- dwelling form to a more complex terrestrial animal. The frog, with its more complex circulatory system, requires a greater se- rum protein concentration from the standpoint of peripheral osmotic exchange (28) and for the maintenance of blood volume (29). Albumin, by virtue of its small molecular weight and its highly charged state at physiological pH’s, is a superior protein for achieving the necessary osmotic balance. Whipple (30) even suggests that the osmotic parameter may itself be stimulatory for the biosynthesis of albumin.

Collateral to the osmotic requirements of the blood is the ne- cessity for greater carrying capacity per unit volume of blood for the small molecules required by the tissues. The transport func- tion of serum proteins and particularly of albumin is well known. An increase in albumin concentration fulfills both the osmotic and the transport needs, resulting in a blood more efficient for meta- bolic exchange in the frog than in the tadpole.

The absence or low concentration of albumin appears to be as- sociated with the presence of a tail in the Anura. In their studies of the serum protein electrophoretic patterns of a series of adult reptiles and amphibia, Dessauer and Fox (16) present a pattern for the salamander Amphiuma means, which they report as typ- ical of five families of Caudata (tailed amphibia or salamanders), a total of 16 species. This pattern is extremely similar to those for the Ranidae tadpole sera, part,icularly to that of the R. heck- scheri tadpole. It is striking indeed, that there is no evidence of a fraction corresponding to albumin in the salamanders. The lack of immunological similarity between tailed and tail-less amphibia, first observed by Nuttall (31) and confirmed by Boyden and Noble (17), could be attributed to this difference. Besides the similarity between the patterns of the Ranidae tadpoles and the adult salamanders which we note, Dessauer and Fox also indicate the close relationship between the patterns of the Ranidae frogs to those of the reptilian order Xauria, the lizards.

Recorded also in Nuttall’s treatise (31)) is the complete lack of serological relationship between the Xenopus frog and the R. temporaria frog. Thus, the observed dissimilarity of the Xeno- pus frog patterns from those of the Ranidae frog is not unex- pected. Nuttall found no evidence of immunological similarities between human serum, Xenopus frog serum, or any of three Ran- idae frog sera. Therefore, while Ranidae frog serum albumin is electrophoretically similar to adult human serum albumin, the two proteins are not immunologically identical.

TI-Thyronine treatment increases the A/G ratio and, for R. catesbeiana and R. heckscheri, the total serum protein in young tadpoles. This is consistent with the observation of a lowering of albumin and total protein after thyroidectomy of rats (32). In R. grylio tadpoles particularly, TI-thyronine may initiate the conversion of globulin protein to albumin as well as reduce the synthesis of serum albumin.

Because of the importance of the liver in the synthesis of serum proteins, the changes in the nature and quantity of the serum proteins that are observed during metamorphosis would be ex- pected to be reflected in the metabolic and structural nature of the liver. Henriques et al. (33) have shown that, in the rabbit, the serum proteins synthesized by the liver represent 40% of the total protein output of this tissue. Kaywin (34) reported extensive changes in the cytology of liver cells during acceler-

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October 1960 A. E. Herner and E. Frieden 2851

ated anuran metamorphosis. An increase in the size and a change in the gross structure of the liver has also been noted (4). Modifications of the metabolic nature of the liver are indicated by the studies of enzymic activities (3-5) and changes in nu- cleic acid metabolism (1).

Extrahepatic changes may account for electrophoretic alter- ations in the -y-globulins and the hemoglobins5 which accompany anuran metamorphosis.

SUMMARY

1. Changes in serum protein concentration and distribution have been studied during the spontaneous and 3,5,3’-triiodo- L-thyronine induced metamorphosis of three species of Ranidae. Comparable changes have been surveyed for the spontaneous development of the species, Xenopus laevis.

2. The most striking change in all the species studied is the increase in and eventual predominance of the albumin fraction. Tadpole sera have extremely low albumin levels. There is no detectable albumin in the serum of some of the Rana heckscheri tadpoles. The progress of anuran metamorphosis is accom- panied by an increase in the total serum protein concentration, accounted for primarily by the marked increase in the albumin concentration.

3. In the Ranidae, metamorphosis results in a redistribution of the globulins in favor of the electrophoretically slower and faster moving components and at the expense of proteins of inter- mediate mobility. The slower moving globulin fractions pre- dominate through out the development of X. Zaevis.

4. The electrophoretic patterns of the young Ranidae tad- poles, particularly R. heckscheri, are similar to those of adult salamanders, whereas patterns prepared from Ranidae frog sera resemble those of the lizards. Of all the species studied, the Xenopus frog patterns are the most similar to human serum pat- terns.

5. Differentiation of the Ranidae patterns ceases with the com- pletion of metamorphosis. However, for the Xenopus frog, the total protein concentration as well as the albumin to globulin ratio continues to increase as the frog grows.

Acknowledgments-We wish to thank Mr. G. Wayne Westmark for his able technical assistance and for his aid in procuring and maintaining the animals. We are indebted to Dr. Harry Lipner for helping us to formulate the bleeding technique. We also should like to acknowledge the contributions of Mr. Lloyd Fish, Mrs. Emma Jo Casson Lewis, and Mr. Thomas P. Bennett to the earlier phases of this work.

5 Changes in the electrophoretic nature of the hemoglobins during anuran metamorphosis will be the subject of a subsequent publication.

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Albert E. Herner and Earl FriedenPROTEINS DURING SPONTANEOUS AND INDUCED METAMORPHOSIS

Biochemistry of Anuran Metamorphosis: VII. CHANGES IN SERUM

1960, 235:2845-2851.J. Biol. Chem. 

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