Hyogo University of Teacher Education Journal Vol. 16, Ser. 3, (1996)

Electrophoretic Analysis of Proteins in the

Regenerating Tail Fin of Goldfish

Megumi KASAHARA* , Akiko MIYAZAKI* * , Motoko OGAWA* ,Akio YAMANOI* * * and Kozo INABA*

(Received September 20, 1995)

Abstract

Changes of proteins in regeneration process of tail fin

of goldfish were analyzed by SDS-polyacrylamide gel

electrophoresis. Protein spectra m supernatant and sedi-

merit fractions were same pattern among tail fin extract

of twenty fish in same body size. There are no differences

on protein pattern among individual goldfish. The degree

of the protein specificity in extract of regenerating tail fin

was expressed by dissimilarity index (D). In supernatant

fraction, D value was 0.14 which was highest 2 weeks at-

ter amputation of tail fin at the distal half, and in sedi-

ment fraction, that was 0.12 and highest 4 or 5 weeks af-

ter amputation. The changes of proteins occured gradual-

ly in regenerating tail fin. Quantitative and qualitative

changes of protein spectra were observed in these super

natant and sediment fractions. In supernatant fraction,

proteins of42 k, 36.5 k and 25.7 k daltons remarkably in-

creased in the early stage of regeneration, and proteins of

68.5 k or 19.5 k daltons increased in the later or middle

stage of the tail fin regeneration, respectively. From

these results, the regeneration process of tail fin ampu-

tated at distal half were discussed from biochemical and

genetical aspects.

Introduction

Regeneration is one of the most important phe-

nomenon of multicellular organisms. It is very compli-

cated and related with cell growth, cell differentiation,

and cell death like apoptosis. Much effort has been ex-

pended to resolve the mechanism underlying process.

The research of this phenomenon has been done by using

newt in the amphibia, planana in the plathelminthes, and

so on. In this work, fish was used because the mechanism

of morphogenesis in fish seems to be more simpler

51

than that in other vertebrate. There are some reports ab-

out the regeneration of the tail fin of fish from the mor-

phological aspect, but as yet no satisfactory conclusion

has been reached (Okada 1943, Hama and Egami 1977).

The biochemical and molecular biological approch to

obtain understanding of the regeneration seems to be

necessary as the next step. In our laboratory, Kawamutsu

(Zacco temmincki) , Silver-Funa (Carassius auratus gibe-

Ho) and goldfish ( Carassius auratus) which may be origin-

ated from Chinese Funa (Carassius auratus auratus)

(Ojima and Takai 1979) have been used for the expen-

ment on the regeneration of tail fin. In this study, the

goldfish was used because keeping of this fish and har-

vesting of regenerating tail fins are very easy, and the re-

generation of the fin seems to be relatively faster than

other fish. An electrophoretic experiment was designed

to try to disclose the substantial bases of the regenera-

tion. The protein or polypeptide spectra were monitored

during the fin regeneration. From these experimental re-

suits, the basic aspects of fin regeneration are discussed.

Materials and Methods

Animals and fin amputation.

Adults of the goldfish, carassius auratus, was used in

this study. Forty goldfishes, which were two years old

and have the body length from 6 to 7 cm, were kept at 25

C in laboratory tanks for a month or more. In all experi-

ments, the distal half of the tail fin was amputated by a

razor blade and after the amputation fish was then kept

at 25 ℃ until the regenerating area harvested for prepa-

ration.

Protein preparation from the regenerating fins.

The regenerating tail fins were collected at 1 week in-

terval after cutting for 16 weeks. These samples were

homogenized in two volumes of phosphate - buffered

Biological Institute, Hyogo University of Teacher Education, Yashiro, Hyogo 673 - 14

日Present address: Hiraoka, Kakogawa, Hyogo 675-01

* H Present address: Hanazono Elementary School, Akashi, Hyogo 673

52

saline (PBS), and centrifuged at 100,000 × g for 60 mm.

The supernatant was decanted into a new tube (super-

natant fraction). After the sediment fraction was

washed two times with PBS, the pellet was resuspended

in PBS and mixed (sediment fraction). Each fraction was

added an equal volume of SDS sample buffer and then

carried out by SDS-polyacrylamide gel electrophoresis.

Protein preparation from the cultured cells and blood.

The fibroblasts-like cells derived from the blastema of

tail fin of goldfish (FBDF) was primary cultured and

further subcultured at 29 ℃. After the cultured FBDF

cells were harvested and then homogenized with two

volumes of PBS, and protein extract was prepared by

centnfuging at 100,000 x g.

The blood of the goldfish was also harvested and the

protein extract was prepared by the method as described

above.

Measurement of the protein content of the regenerating

fins.

The protein content of supernatant fraction of the re-

generating tail fin was measured by Lowry method

(1951). Bovine albumin was used as standard protein.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

(SDS-PAGE).

SDS-PAGE of the sample was carried out using the

method of Laemmli (1970). The equal amount of protein

(SO ^g) in each sample was electrophoresed. The spacer

gel was 4 % polyacrylamide and the separation gel, 15

%. For the detail analysis, SDS-PAGE was performed at

6 %, 9 % and 12 % of the gelconcentration. 45 mMTns-

glycine (pH 8.3) was used as running buffer. After elec-

trophoresis the gel was stained with Coomassie brelhant

blue R-250.

Porcine collagen was used as standard protein in SDS-

PAGE of sediment fraction. Effect of blood protein in

the sample were compensated on the protein analysis by

SDS-PAGE.

Results

Morphological changes in the regeneration process of tail

fin.

The goldfish whose tail fin was amputated at the distal

half of fin by a razor blade was kept at 23-25 ℃ until the

fin was regenerated to its normal size. For the external

morphology, this process takes about 4 weeks in the good

condition but the pigment of tail fin is not yet repro-

duced. The weight of the regenerating tail fin is reco-

(当IBA3APBPH)一息p/A

000a

o

r

^

¥

o

12 16

Time (week)

Fig. 1. Relative weight of the regenerating tail fin of goldfish

as compared with that of normal tail fin amputated at

distal half.

vered for 8 weeks (Figure 1). These results suggest that it

takes at least 8 weeks for the regeneration of tail fin in

the protein recovery and morphological aspect.

The general pattern of proteins among individual tail fin

of goldfish.

The protein spectra of the normal tail fin and the re-

generating fin of goldfish were analyzed by SDS-PAGE.

Twenty fishes of the same body size were examined. In

normal tail fin, the protein spectra of the supernatant and

sediment fractions were same pattern among twenty fish

(data not shown). In regenerating process, the similar

results were observed (Figure 2). These results indicate

that there are no differences of the protein spectra

among twenty goldfish. Therefore, following experi-

ments were done by harvesting the regenerating tail fins

of two or three fishes.

Protein spectra in the regeneration process of tail fin.

Electrophoretic analysis of protein specificity of the re-

generating tail fin was done in order to assess the genetic-

al basis of the regeneration. In normal fin, 76 bands of

polypeptide were observed in the supernatant fraction

and 52 bands in the sediment fraction. In the regenerat-

ing tail fin, there are differences in number of

Electrophoretic Analysis of Proteins in the Regenerating Tail Fin of the Gold丘sh

Fig. 2. The protein spectra of the supernatant fractions derived from tail fins which were amputated at distal half of individual

goldfish. In each picture, lane numbers indicate the time (weeks) at which the regenerating tail fin was amputated. Symbol

N indicates the normal tail fin. Symbol B indicates the blood of the goldfish. oM" indicates the molecular weight markers

whose weights are shown in daltons on the left margin. No significant differences in these supernatant fractions are

observed at the same time of amputation. In the sediment fraction, no differences are observed as same as the case of su-

pernatant fraction (data not shown). Sup., supernatant fraction; Sed., sediment fraction

Table 1. Distribusion of protein bands in the regeneration processes of tail fin by SDS-PAGE. The differences in protein bands

were expressed as dissimilarity indices (D). The protein bands in regeneration processes of tail fin were compared with

those in normal tail fin amputated at distal half.

Regenerationtime normal 1 week 2weeks 3weeks 4weeks 5weeks 6weeks 7weeks 8weeks 12weeks 16weeks

Weight (Relative value) (1∝I%) (28%) (50%) (64%) (75%) (84%) (95%) (99%) (1(刀(1∝)%) (1∝I%)

Total pepti de bands Sup. 76 77 77 77 76 76 76 77 77 76 77

Sed. 52 52 52 52 52 50 52 52 52 52 52

Sup.+Sed. 128 129 129 129 128 126 1 28 129 129 128 129

Number of different Sup.

bands in quality (a) Sed.

Sup.+Sed.

101

303

224 I

O

l

000

Number of different Sup.

bandsinquantity (b) Sed.

Sup.十Sed;

o

o

T

f

2

o

o

蝣

*

"

ioin2

r

-

サ

2

5

4

q

ノ

347

o

)

蝣

*

¥

o

246

245

246

Number of total Sup.

different bands (a+b) Sed.

Sup.+Sed. 13

‖

4

1

5

ovu")3

961 t

^

*

0

2

監S^K'山

347

347 細

5^b.'>:

347

Dissimilarity index (D) Sup. 0.12 0.14 0.12

Sed. 0.08 0.08 0. 10

Sup.+Sed. 0.10 0.12 0. 1 1

川畑.0

0

0

0

心*'&

0

0

0

加欄.0

0

0

0

朋個.0

0

0

0

げ欄r6

0

0

0

個tso

000

222

I

l

l

0

0

0

sup., supernatant fraction; sed., sediment fraction.

54

Table 2. Distnbusion of protein bands separated by SDS-PAGE and dissimilarity indices (D) of protein bands in the regeneration

processes of the tail fin.

Regenerationperiod normal- 1 1-2 2-3 3-4 4-5 5-6 6-7 7-8 8-12 12-16 16-normal

(week)

Number of different Sup.

bands in quality (a) Sed.

Sup.+Sed.

_

0

-

000

000 1

0

l

000

202 l

O

l

I

O

l

101

Number of different Sup.

bandsin quantity (b) Sed.

Sup.+Sed. 12

224

549 f

i^^U巴F<

4

2

′

0

224

202

202

Number of total Sup.

different bands (a+b) Sed.

Sup.+Sed. 13

224

5

4

0

ノ

527 4

2

!

b

t

N

ォ

303

325

202

<

M

蝣

*

V

O

4

2

′

0

ヽ

蝣

n

o

r

*

-

.

347

Dissimilarity index (D) Sup. 0.12

Sed. 0.08

Sup.+Sed. 0. 10

潤-S

000

仙

」

=

・

0

0

0

3.'C1

0

0

0

昭

的

皿

0

0

0

仙㈱1-:

0

0

0

0

5

糾

0

5

0

0

0

'ff川、

0

0

0

那

加

8

0

0

0

鵬

悌

o

0

0

0

王}舶用

ハ

U

O

O

sup., supernatant fraction; sed., sediment fraction.

Sup.

Fig. 3. The protein spectra of regenerating tail fins of goldfishes by SDS-PAGE. Panels Sup. and Sed. indicate the supernatant

and sediment fractions, respectively- All symbols are

polypeptide bands at least 1 or 2. It indicates that these

bands are different in quality. The other bands were

commonly found in each stage of the regenerating tail

fin. These results are shown in Figure 3 and Table 1.

The degree of the protein specificity in extract of re-

generating tail fin was the expressed by a similarity index

(S) or dissimilarity index (D) defined as follows (Odum

1971):

S-2C/(A+B)

D-l-S

the same as those of the Fig. 2.

where A is the sum of number of bands in both super-

natant and sediment fractions of normal tail fin, B is that

of the regenerating tail fin, and C is the number of bands

commonly found in normal and regenerating tail fins.

D value between normal tail fin and regenerating tail

fin which was harvested 1 week after amputation was

0.12 in supernatant fraction (Table 1). This value is high-

est 2 weeks after amputation. In the sediment fraction, D

value was highest 4 or 5 weeks after amputation, and it

was 0.12. In the total D value, the difference between the

normal and regenerating tail fin is higher in early stage

Electrophoretic Analysis of Proteins in the Regenerating Tail Fin of the Goldfish

Regene.-8 Regene.- 12

55

Fig. 4. Schematic representation of protein spectra in the regenerating tail fin. Panels A and B indicate the supernatant and sedi-

ment fractions, respectively. The protein bands which pronouncely changed in the regenerating tail fin are shown by bold

lines. The width of band indicates the relative quantity of protein. The molecular weight of specific bands in the regenera-

tion process of tail fin are indicated on the right margin. For the explanation of symbols, shown in Fig. 2.

56

than later one of tail fin regeneration. These results indi-

cate that apploximaly 12 % of the genes of the total

genome must be turned on or off to cause regeneration of

tail fin. Distribution of protein bands and D values in the

regeneration process of tail fin was shown in Table 2.

These data indicate that the peak ofD value was 3 weeks

after amputation, and later it decrease, and that the

changes of protein in regenerating tail fin gradually

occured.

In the regeneration process of tail fin, the qualitative

change of protein was scarcely observed, but the quan-

titative change was detected. SDS-PAGE of polypeptides

derived from regenerating tail fin was shown in Figure 3,

and its results were summarized in Figure 4. Proteins of

42 k, 36.5 k and 25.7 k daltons remarkably increased in

the supernatant fraction derived from regenerating tail

fin in early stage 1 to 4 weeks after amputation (Figure

3A and Figure 4A). Proteins of68.5 k and 19.5 k daltons

increased in the supernatant fraction derived from re-

generating tail fin in the later or middle stage, respective-

ly. In the cultured FBDF cells a specific polypeptide

band of 18.6 k dalton was detected in their supernatant

fraction (data not shown).

Quantitative changes of proteins were also observed in

the sediment fraction derived from regenerating tail fin.

These results shown in Figure 3B and Figure 4B. It seems

that two polypeptide bands over 90 k daltons in the sedi-

ment fraction may be collagen, since these bands

accorded with the position of standard collagen in SDS-

PAGE (data not shown). From D value and polypeptide

pattern, we conclued that quantitative changes of pro-

teins in the regeneration process of tail fin occured re-

markably 2 or 3 weeks after amputation.

Discusion

The changes of protein patterns in the regenerating tail

fin of goldfish has been examined by SDS-PAGE. The

tail fin which was amputated at the distal half was re-

generated to its normal size for 8 weeks, but this regen-

eration period was not enough in the recovery of protein

or polypeptide level of regenerating tail fin. In our study,

the polypeptide spectra was monitored for 16 weeks after

amputation of the tail fin, but its polypeptide pattern was

not recovered completely to its initial pattern. On this

point, more resarch is needed. D values show that the

changes in protein level occured gradually, and 3 weeks

after the amputation of normal tail fin at the distal half

those were the most changeable. Furthermore it seems

that the genes at least 12 % was related with the regen-

eration of tail fin. From polypeptide pattern, there are

two types of protein expression in tail fin regeneration.

First type expresses early stage and mainly in supernatant

fraction, and regulatory proteins associated with the re-

generation process, like growth facters, may be express-

ed. Second type expresses in later stage and mainly in the

sediment fraction derived from regenerating tail fin.

These proteins are increased gradually, and it seems that

they are structure proteins, like muscle actin and col-

lagen. There is a report on the protein pattern of the fish

muscle by SDS-PAGE (Childs 1973). In this paper, at

least 10 actin proteins were detected. These proteins may

be also included in the regenerating tail fin of goldfish,

and concentrated polypeptide bands -detected by SDS-

PAGE may be similar to muscle proteins. From another

aspect, the surface of tail fin which was injured a little be-

came black, but the tail fin amputated at the distal half

regenerate to white tail fin without melanophores. It

reasoned that the regeneration of black tail fin was reco-

vered to initial state, and in the white tail fin the protein

pattern was not recovered completely and need more

long periods for the complete regeneration of tail fin.

Further works are necessary to elucidate the detail of

protein expression in the regenerating tail fin.

Refe rence

Childs, E. A. 1973. Electrophoresis of fish myofibnllar pro-

teins with sodium-dodecyl sulfate-polyacrylamide gel. J.

Fish. Res. Board Can. 30: 706-707

Hama, A., and N. Egami. 1977. The dose-rate effect of γ-

irradiation on the initiation of mitosis in the regenerating

tail fin of the fish Oryzias latipes. J. Fac. Sci. Univ. Tokyo

14: 47-60.

Laemmili, U. K. 1970. Cleavage of structural proteins during

the assembly of the head of bactenophage T4. Nature 277:

680-685.

Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Ran-

dall. 1951. Protein measurement with the folin phenol rea-

gent. J. Biol. Chem. 193: 265-275.

Odum, E. P. 1971. "Fundamentals of Ecology , Saunders

Company, Philadelphia, p.574

Ojima, Y., and A. Takai. 1979. Further cytogenetical studies

on the origin of the gold - fish. Proc. Japan Acad. 55: 346-

350.

Okada, Y. 1943. Regeneration of the tail in丘sh. Annot. Zool.

Japon. 22: 59-69.