Effects of hormones on tail regeneration and regression in Rana catesbeiana tadpoles

Effects of hormones on tail regeneration and regression in Rana catesbeiana tadpoles

GENERAL AND COMPARATIVE ENDOCRINOLOGY 29, 376-382 (1976) Effects of Hormones on Tail Regeneration and Regression in Rana catesbeiana Tadpoles CHE...

2MB Sizes 0 Downloads 13 Views

GENERAL

AND

COMPARATIVE

ENDOCRINOLOGY

29, 376-382 (1976)

Effects of Hormones on Tail Regeneration and Regression in Rana catesbeiana Tadpoles CHEUK W. LI’

AND HOWARD A. BERN

Department of Electrical Engineering and Zoology and Cancer Research Berkeley, Accepted

Electronic Research Laboratory; Department of Laboratory, University of California, California 94720 March 12, 1976

Thyroxin inhibits tail regeneration of Rana catesbeiana tadpoles and induces tissue resorption in the tail regenerates as tadpoles approach late metamorphic stages. The resorption of regenerative tissue appears to require a critical concentration of thyroxin at a particular metamorphic stage. F’rolactin, the “larval growth hormone,” did not stimulate growth of the regenerating portion of the tail. Growth hormone, however, stimulated tail regeneration during the initiation of young tissue growth and maintained the rate of growth at later stages. Neither prolactin nor growth hormone significantly antagonized the thyroxininduced regression of the regenerating tissue.

There is increasing evidence that the control of metamorphosis in amphibians is bihormonal, involving not only thyroxin but also prolactin (Etkin, 1968; Bern and Nicoll, 1968; Frye et al., 1972; Bern, 1975). Prolactin appears to act antagonistically to thyroxin and stimulate growth in the bullfrog tadpole (Bern et al., 1967; Etkin and Gona, 1967; Gona, 1967). The height and length of the tail increase after administration of even small amounts of prolactin. Etkin (1968) suggested that there is an interaction between thyroxin and prolactin during metamorphosis. During early premetamorphosis, prolactin dominates and stimulates body growth. During metamorphic climax, thyroxin dominates as prolactin release is greatly reduced, resulting in tail resorption and eventually completion of the adult form. Mammalian growth hormone may also be slightly effective in stimulating growth (Bern et al., 1967). One of the goals of the present study is to determine whether hormones affect regenerative growth of the r Present address: Department of Otorhinolaryngology, Albert Einstein College of Medicine, Bronx, N. Y. 10461.

tail in the same manner as they affect its normal growth. Amputation of the tail tips from lizards, urodeles, and anuran tadpoles results in regeneration at the cut edges (Speidel, 1947; Rose, 1964; Simpson, 1965; Licht and Howe, 1969; Vethamany, 1970). Licht and Howe (1969) found that tail regeneration in the adult lizard Anolis depends on a combination of adenohypophyseal hormones (growth hormone, prolactin, gonadotropin, and thyrotropin). Prolactin stimulates the size of the regenerating tails, but appears not to affect the rate of blastema formation and of tail elongation (Licht and Jones, 1967). Hormonal dependence of tail regeneration in adult newts (Diemictylus viridescens) has also been reported (Vethamany-Globus and Liversage, 1973). Insulin, growth hormone, and thyroxin individually stimulate growth and differentiation of the explanted newt tail blastema. However, a combination of these hormones together with cortisol results in maximum growth and advanced differentiation of the explants. Thyroxin influence on tail regeneration of anuran tadpoles has also been demonstrated. Weber (1967) showed that if 376

Copyright All rights

@ 1976 by Academic Press, Inc. of reproduction in any form reserved.

HORMONES

AND

TADPOLE

tail regeneration in Xenopus is initiated shortly before metamorphic climax, the newly formed regenerate resists the regressive influence of thyroxin longer than the tail stump does. Chou and Kollros (1973) suggested that the difference is due to differential tissue sensitivity to thyroxin. The tail regenerate appears to exhibit characteristics of young tissue rather than that of tissue dose to metamorphic climax. However, growth hormone, which stimulates tail regeneration in both lizards and newts, has not been examined for effects on tail regeneration in anuran tadpoles. Furthermore, prolactin, which may be the “growth hormone” of anuran tadpoles (Rana catesbeiana) (Bern et al., 1967)and which stimulates tail regeneration in Anolis (Licht and Jones, 1967), has not been studied in regard to anuran tadpole tail regeneration. MATERIALS

AND METHODS

In the first series of experiments, tadpoles of the bullfrog Rana catesbeiana were separated into four groups. Each group contained equal numbers of tadpoles of comparable metamorphic stages. Every second day for 17 days, tadpoles were injected intraperitoneally with ovine prolactin (oPRL: NIH-PSO), bovine growth hormone (bGH: NIH-GH-B8), or 0.01 N NaOH in volumes of 25 ~1. Both oPRL and bCiH were dissolved in 0.01 N NaOH at a concentration of 1 &PI. Each animal was maintained in a separate plastic box in approximately 300 ml of tap water. With the exception of the last group of tadpoles (group 4), the water contained thyroxin at a concentration of 0.025 &ml. Aquarium water was changed at the time of each injection, and a halfspoonful of canned spinach was added as food. On Day 1, each tadpole was staged (Rugh, 1%2), and tail height and tail length were measured with calipers. About 7 mm of tail tip were amputated, and the tail length was measured again. On every fourth day, starting on Day 5, tail height and tail length were again measured. On Days 9, 13, and 17 the tail regenerate of each animal was examined under a dissection microscope, and the outline of the regenerating tail fin was traced on graph paper; the length and the area of the regenerate were then determined. The second experimental series was carried out in the same manner as the first, with the addition of two groups (5 and 6). Group 5 received 25 pg of bGH in volumes of 25 J every 2 days, and group 6 received 25 pg of oPRL similarly. The concentration of

TAIL

REGENERATION

377

thyroxin in tap water for groups 4,5, and 6 was lowered to 0.015 j&nl. The third experimental series was repeated in the same manner as the second series. However, thyroxin concentration was increased to 0.025 &ml. The animals in the first experiment were taken from a different pond from that supplying tadpoles for the last two experiments. The average tadpole weight in the last two experiments was about 20 g, about two to three times greater than that of the tadpoles used in the first experiment.

RESULTS

The tail stump of Runu cutesbeiunu tadpoles regenerates normal structures, including lateral-line sensory placodes (Fig. 1; cf. Li, 1975), as was reported by Speidel (1947, 1948) in Runu clumituns. The areas of the regenerating tail fins of all tadpoles (both control and hormonetreated) continued to increase during the period of hormone treatment with the exception of tadpoles treated with thyroxin alone or in combination with other hormones, in Experiment 1 (group 4) and Experiment 3 (groups 4-6) (Figs. 1A-C). Growth hormone appeared to stimulate tail regeneration significantly in Experiments 2 and 3 (Table 1). In Experiments 1 and 3, the high dose of thyroxin (0.025 @g/ml) alone significantly inhibited tail regeneration and initiated tissue resorption in the regenerating fins after tail regenerates attained a certain size. The lower thyroxin treatment in Experiment 2 appeared to have no inhibitory effect on tail regenerates and no influence on tissue resorption in the regenerating fins. Prolactin failed to have any significant effect on tail regeneration. Based on the Duncan multiple range test, GH and PRL show no antithyroxin activity on tail resorption in the regenerating tissue (Table 1). In all experiments (Table 2), the control tadpoles showed no significant changes in overall tail length and tail height, whereas tadpoles treated alone with PRL or GH showed significant increase in tail height and tail length throughout the experiments (cf. Bern er al., 1967; Li, 1975). In Experi-

FIGURE

378

1

HORMONES

AND TADPOLE

TAIL

379

REGENERATION

TABLE 1 RESPONSE OF REGENERATING BULLFROG TADPOLE TAIL TO HORMONES (AT DAY 17) Experiment

Group number

Hormonal treatment

Number of Tail regenerate taduoles area (mm*)

1

1 2 3 4

Control Prolactin Growth hormone Thyroxin

14 13 11 6

8.21 6.69 8.91 3.75

2

1 2 3 4 5 6

Control Prolactin Growth hormone Thyroxin Thyroxin + prolactin Thyroxin + growth hormone

9 10 12 13 6 10

7.67 11.55 16.08 8.19 14.67 10.00

+- 0.86 2 1.07 -’ 1.25 ‘- 1.16 k 6.12 -, 1.09

1 2 3 4 5 6

Control Prolactin Growth hormone Thyroxin Thyroxin + prolactin Thyroxin + growth hormone

8 11 10 10 7 7

6.44 7.64 10.70 2.70 4.29 4.14

5 0.86 -r- 0.95 k 1.04 f 0.43 f 0.49 k 0.46

3

2 -t ” 2

0.64 0.69 0.86 0.94

P

<0.01* <0.01* 0.01
< 0.05**

0.01


* Compared with control (Dunnett test). ** Compared with prolactin (Duncan multiple range test). *** Compared with growth hormone (Duncan multiple range test).

merits 1 and 3, tadpoles treated alone with higher amounts of thyroxin showed significant decrease in tail length and tail height by the end of the experiments. In Experiment 2, tadpoles with a lower amount of thyroxin alone showed continuous increase in tail length; however, by the end of the experiment, tail height had decreased significantly. The tail height and tail length of tadpoles treated with both thyroxin and PRL or with both thyroxin and GH decreased significantly throughout the experiment. However, in Experiment 3, tail height and tail length of tadpoles treated with both thyroxin and PRL or with both thyroxin and GH increased significantly only in the early days of the experiment. Toward the end of the experiment, the tail

length and tail height of the tadpoles treated with both thyroxin and prolactin decreased to the control level. Tadpoles treated with both thyroxin and GH showed tail height in the control range, although tail length remained significantly greater than in controls . At the beginning of the experiments, the tadpoles were staged at a range of stages 2 to 11. On Day 11 of the experiments, the control tadpoles and those treated with PRL or GH showed a slow rate of metamorphosis attaining stages not more advanced than stage 12. On the other hand, tadpoles treated with thyroxin alone or in combination with other hormones attained more advanced metamorphic stages (19-21) (Li, 1975).

FIG. 1. (A) Light micrograph of tail tip from stage 8 tadpole after regeneration for 17 days. Arrowheads indicate placode formation in the lateral-line organ. (B) Scanning electron micrograph of tail regenerate from stage 8 tadpole. Arrowheads indicate the newly formed lateral-line organs. (C) High magnification of newly formed lateral-line organs. Cupula of the sensory structure was removed to show individual hair cells. (D) Lateral-line organ from intact tail. Cupula was removed, revealing several hair cells.

380

LI

AND

BERN

TABLE 2 RELATIVE

Experiment

Group number

RESPONSE

OF BULLFROG

Hormonal treatment

TADFQLE

Number of tadpoles per group

TAILS

TO VARIOUS

HORMONE

Percent change from initial tail height

Percent change from initial tail length

Day 17

Day9

TREATMENTS

Day 9

Day 17

1

1 2 3 4

Control Prolactin Growth hormone Thyroxin

14 13 11 6

2.4 30.2 18.8 - 0.2

2 5 f f

1.5 2.4** 2.3** 2.8

2.6 38.8 23.5 -39.8

k 2 f f

1.8 2.9** 2.0** 5.9**

5.7 18.3 15.6 9.6

-’ t 2 2

1.0 2.S** 1.2** 2.3

10.1 25.5 24.1 - 9.5

% ‘t T

1.3 3.W’ 1.8** 6.7**

2

1 2 3 4 5

Control Prolactin Growth hormone Tbyroxin Thyroxin + prolactin Thyroxin + growth hormone

9 10 12 13 6

4.5 20.7 22.0 3.1 18.2

-r f f tf

2.7 2.4** 1.8** 0.8 2.7**

7.2 33.8 37.0 - 7.4 23.0

f 2 5 i k

2.5 2.5** 2.8** 1.1** 5.7**

- 0.5 4.6 7.2 3.5 6.7

2 -’ 2 2 -’

0.5 1.3** O.S** 0.8* 1.4**

3.9 11.8 19.7 10.0 18.5

2 f 2 2 +

0.7 2.2* 1.5** 1.3 4.3**

10

15.3 f 1.6**

6 3

* Signiticantly ** Siiilicantly

Control Prolactin Growth hormone Thyroxin Thyroxin + prolactin Thyroxin + growth hormone different different

from control; from control;

8 11 10 10 7 7

using Dunnett using Dunnett

4.8 22.7 20.0 - 1.2 18.3

f f k 2 f

1.5 1.7** 2.8** 1.7 2.2**

16.4 k 2.9**

17.7 5 2.8* 3.8 2 1.6 30.1 z!z 1.9** 29.3 -’ 2.9** -29.6 -t 2.8** - 1.6~ 3.9 - 4.0 f 4.0

6.8 2 1.3** -

1.8 10.2 9.8 4.6 14.8

2 2 f I! 2

1.8 2.2** 3.6* 1.8 3.4**

15.4 2 3.6**

21.8 t 3.2** 3.6 25.0 18.8 - 10.4 3.2

f k 2 5 2

2.8 3.4** 3.8’ 5.6* 8.4

18.8 2 1.6

test, P c 0.05. test, P < 0.01.

DISCUSSION Thyroxin induces tail regression in anuran tadpoles. The data obtained in these experiments suggest that the hormone also inhibits tail tin regeneration and induces tissue resorption in tail regenerates as tadpoles approach late metamorphic stages. Furthermore, our results favor the concept of differential tissue threshold sensitivity to thyroid hormone at different metamorphic stages (Kollros, 1961). Tail regenerates continued to grow in premetamorphic and prometamorphic stages, even under the influence of a high concentration of thyroxin. However, at or near metamorphic climax, regeneration was inhibited, and the regenerates began to regress. Concentration of circulating thyroxin is critical in tail regression (E&in, 1964; Derby, 1968). Apparently, this is also true for the regression of the regenerating tail fin. Exposure to lower amounts of thyroxin in the second experi-

ment resulted in no sign of regression, whereas higher amounts induced resorption. In summary, it appears that resorption of regenerating tail tissue requires a critical concentration of thyroxin at a particular metamorphic stage. Prolactin, the proposed “larval growth hormone” in amphibians (Bern et al., 1967; Etkin and Gona, 1967), increased height and length of the tail fin even at a low dosage. However, in all our three experiments, it did not stimulate growth in the regenerating portion of the tail. It also did not antagonize the thyroxin-induced regression during regeneration. On the other hand, growth hormone, possibly favoring development of adult structures (e.g., development of limbs; Bern er al., 1967) during metamorphic climax, distinctly stimulated growth of the regenerating tadpole tail tip. Chou and Kollros (1973) found that the tail regenerate exhibits the properties of

HORMONES

AND

TADPOLE

very young fin tissue, which resists thyroid-induced regression longer than the intact tail fin. In conclusion, growth hormone, similar to the role of prolactin in the growth of intact tail fin and of the body of stimulates and maintains the tadpole, growth of regenerating amphibian tissue. Unlike prolactin, growth hormone does not antagonize the thyroxin-induced regression of the intact tail. It is possible that the regenerating tissue is not sensitive to prolactin, whereas it is sensitive to growth hormone. As the regenerating tissue ages, it may become sensitive to the influence of prolactin as is the intact tail tissue. It has also been shown that growth hormone stimulates tail regeneration in the reptile (Licht and Howe, 1969) and the urodele (Vethamany-Globus and Liversage, 1973). Our results indicate that growth hormone stimulates tail regeneration during the initiation of young tissue growth in the larval Rana catesbeiana and that both prolactin and growth hormone maintain the rate of growth at later stages. Somatotropic effects of mammalian growth hormone on the larval newt (Taricha torosa) were reported in both early and late larval periods (Licht et al., 1972). Similar effects were also observed in the larval Rana catesbeiana (in Bern et al., 1967, see Fig. 3). It appears that growth hormone, which is present in the pituitary of larval Rana catesbeiana (Clemons, 1971), may serve as a growth factor for both normal and regenerative development of the bullfrog tadpole tail.

TAIL

Bern, H. A., and Nicoll, C. S. (1968). The comparative endocrinology of prolactin. Rec. Progr. Horm. Res. 24, 681-720. Bern, H. A., Nicoll, C. S., and Strohman, R. C. (1967). Prolactin and tadpole growth. Proc. Sot. Exp. Biol. Med. 126, 518-521. Chou, H. I., and Kollros, J. J. (1973). Regression in regenerated tails of Rann pipiens. Amer. Zool. 13, 1351. Clemons, G. K. (1971). “Changes in Growth Hormone and Prolactin Levels in the Pituitary of Frogs during Various Physiological and Developmental States.” M.A. Thesis in Physiology, University of California, Berkeley, Calif. Derby, A. (1968). An in virro quantitative analysis of the response of tadpole tissue to thyroxine. J. Exp.

Zool.

This research was aided by National Institutes of Health Grant CA-05388 to the Cancer Research Laboratory, and Grant GM-17523-03 to the Electronic Research Laboratory, University of California, Berkeley. We are indebted to Dr. Joe W. Crim and Professor E. R. Lewis for their suggestions. oPRL and bGH were generously supplied by NIH.

REFERENCES Bern, H. A. (1975). On two possible primary activities of prolactins: Osmoregulatory and developmental. Verh. Dew. Zool. Gesell. 1975, 40-46.

168, 147-156.

Etkin, W. (1968). Hormonal control of amphibian metamorphosis. 1r1 “Metamorphosis” (W. Etkin and L. I. Gilbert, eds.), pp. 313-348. AppletonCentury-Crofts, New York. Etkin, W., and Gona, A. (1967). Antagonism between prolactin and thyroid hormone in amphibian development. J. Exp. Zoo/. 165, 249-258. Frye, B. E., Brown, B. S., and Snyder, B. W. (1972). Effects of prolactin and somatotropin on growth and metamorphosis of amphibians. Gen. Comp. Endocrinol.

Suppl.

3, 209-220.

Gona, A. (1967). Prolactin as a goitrogenic agent in amphibia. Endocrinology 81, 748-754. Kollros, J. J. (1961). Mechanisms of amphibian metamorphosis. Amer. Zool. 1, 107-114. Li, C. W. (1975). “Structure and Development of Hair Cells in the Acoustico-lateralis System of the Bullfrog.” Ph.D. Thesis in Electrical Engineering, University of California, Berkeley, Calif. Licht, P., Cohen, D. C., and Bern, H. A. (1972). Somatotropic effects of mammalian growth hormone and prolactin in larval newts, Turicha torosa.

Gen.

Comp.

Endocrinol.

18, 391-394.

Licht, P., and Howe, N. R. (1969). Hormonal dependence of tail regeneration in the lizard Anolis carolinensis.

ACKNOWLEDGMENTS

381

REGENERATION

J. Exp.

Zool.

171, 75-84.

Licht, P., and Jones, R. E. (1967). Effects of exogenous prolactin on reproduction and growth in adult males of the lizard Anolis carolinensis. Gen.

Comp.

Endocrinol.

8, 228-244.

Rose, S. M. (1964). Regeneration. In “Physiology of the Amphibia” (J. A. Moore, ed.), pp. 545-622. Academic Press, New York. Rugh R. (1962) . “Experimental Embryology,” pp. 70-74. Burgess, Minneapolis. Simpson, S. B., Jr. (1965). Regeneration of the lizard tail. In “Proceedings. Regeneration in Animals,” pp. 431-443. North-Holland, Amsterdam. Speidel, C. C. (1947). Correlated studies of sense organs and nerves of the lateral line in living frog

382

LI AND BERN tadpoles. I. Regeneration of denervated organs. J. Comp. Neurol. 87, B-55.

Speidel, C. C. (1948). Correlated studies of sense organs and nerves of lateral line in living frog tadpoles. II. The trophic observations of specific nerve supply as revealed by prolonged observations of denervated and reinnervated organs. Amer. J. Anat. 82, 271-320. Vethamany, S. (1970). “‘Zn Vivo and in Vitro Studies on the Influence of Hormones on Limb and Tail

Regeneration in Adult Diemictylus viridescens. ” Ph.D. Thesis in Zoology, University of Toronto, Toronto. Vethamany-Globus, S., and Liversage, R. A. (1973). In vivo studies of the influence of hormones on tail regeneration in adult Diemictylus viridescens. J. Embryol. Exp. Morphol. 39, 397-413. Weber, R. (1967). Biochemistry of amphibian metamorphosis. In “The Biochemistrv of Animal Development” (R. Weber, ed.), pp. 227-302. Academic Press, New York.