Action of human placental lactogen on second metamorphosis in the newt Notophthalmus viridescens

GENERAL

AND

Action

COMPARATIVE

of

21, 377-380 (1973)

ENUOCRINOLOGY

Human

Placental

in the

Newt

School,

GONA

AND

of Biology, Montdnir 070@; Department iO9

on Second

Nofophfhalmus

OPHELI.4 Departmeld New Jersey

Lactog.en

Rergen

Metamorphosis

viridescens

AMOS G. GONA

Slate College, Upper Moutclui,~. of Anatomy, New Jersey Medical Newark, New Jersey 0110.7

Street,

Received January 30, 1973 The effects of human placental lactogen (HPL) on metamorphosis were studied in red efts (land stage) of the newt Notophthalmus viridescew Three groups received t,he following injections: (i) low (10 pg) doses of HPL; (ii) high (100 fig) doses of HPL; (iii) 10~ doses of HPL plus 0.01 pg thyroxine (T4). The low dosage level was most effective in inducing migration into water (water drive) and increasing the tail height.. The higher .dose was least effective in inducing water drive and had no effect on tail height. No synergistic effect was noted with HPL plus T, treat.ment. Thus, HPL has a clear-cut prolactin-like effect in that both substances can induce second metamorphosis. However, HPL differs strikingly from prolactin in the failure of high doses to induce water drive and also in the absence of synergism with T,.

The fascinating phenomenon of metamorphosis in the red-spotted newt, Notophthalmus viridescens, has been the subject of several studies (see Grant, 1961). The aquatic larva, like the frog tadpole, metamorphoses under the influence of thyroid hormone into a land stage. After about three years of terrestrial life, the land stage (red eft) metamorphoses into the adult stage, which migrates back into water. The phenomenon of second metamorphosis (water-drive phenomenon) was discovered by Chadwick (1940) to be under the control of pituitary prolactin. No other hormone is known to induce second metamorphosis. In view of t,his specificity, it is conceivable that the red eft can be used as a t’est animal to test for prolactinlikc action of any given substance. Human placental lactogen (HPL), also known as human chorionic somatotropin, is of special interest in this connection since it is described as possessing properties of both prolactin and growth hormone by various tests (Josimovich and MacLearen, 1962; Grumbacb et al.. 1968; Sherwood, 1969;

Beck, 1972), but to our knowledge has not been examined in the red eft test. Our previous work had demonstrated synergism between prolactin and low levels of T, in the red cft, although high levels of T, were found to inhibit water drive (Gona et al., 1970, 1973). In the present work we explored the possibility of synergism between T, and HPL. The choice of dosages in this study was based on our experience with prolactin and T, in the earlier work. MATERIALS

AND

METHODS

Red efts (terrestria.1 stage) of the newt No0.9-1.0 g. were collected in Massachusetts. Four groups of 10 animals each were injected intraperitoneally on alternate days as follows: Group 1, 100 pg HPL; Group 2, 10 gg HPI,: Group 3, 10 pg HPL and 0.01 pg thyroxine (T,) ; Group 4, 20 kI of the

tophthnlm~rsviridescens, weighing

qalinc ,,ehicle, L The HPL (Nut.ritional

Biochemicals Corp.) was dimolved in 0.66% saline. Thyroxine (thyroxine sodium pentahydrate, Mann Research Laboratory) was dissolved in a few drops of 0.1 N sodium hydroxide and then diluted with 0.66% 377

Copyright @ 1973 by Academic Press. Inc. All rights of reproduction in any form reserved.

378

GONA

AND

GONA

saline. Intraperitoneal injections were administered laterally, immediately rostra1 to the hind 1iml)s. Hamilton microsyringes with fixed 30gauge needles were used. The animals were kept in transparent plaslic dishes containing a bc’d of moss and a dish of water at one end (see Gona et al., 1970). At the end of the experiment (20 days), tail I ips were collected and the height was measured at, a point 2.5 mm from the tip. For this mrasuremvnt, a Bausch and Lomb microprojector was usr~l with the magnification adjust4 to 10. RESULTS DAYS AFTER FIRST INJECTlOP<

Skin Changes The control animals (Group 4) retained their rough, dry skin. The animals of the other three groups began to show signs of integumentary changes indicative of second metamorphosis in about 4 days. At first these changes were distinctly more prominent in the group injected with the higher (100 klg) dose of HPL. But soon the skin of all animals in these three experimental groups turned moist, smooth, and sticky. The skin color began to change from redtlish orange to light olive green. ll’ater

Drive

Spontaneous migration into water was first noticed after 9 days of treatment. The animals injected with 10 pg HPL (Group 2) showed the most pronounced water drive effect. By day 19, 9 of the 10 animals in this group entered water. Of the three hormone-treated groups, Group 1 animals (100 pg HPL) showed the least tendency to enter water spontaneously. Most of the time none of the animals in this group were found in water. When placed in water, nlmost, all of them crawled out within a few minutes. Supplementation of 10 /*g HPL by 0.01 pg T, did not increase water drive, but rather had an inhibitory effect. Details of the spontaneous water drive effect for the different experimental groups are given in Fig. 1. None of the control (Group 4) animals entered water. Tail Height Compared with controls (Group 4) the tail height showed a significant increase

FIG. 1. Effects of (HPL) and thyroxine red cfts.

human (T,)

placental on water

lactogen drive in

only in the animals which received 10 ,pg HPL (Table 1). Although the data are not sufficient for statist,ical evaluation of the possibility of an inhibitory effect, it is clear that the supplementation of HPL by T, did not augment the effect. of HPL. DISCUSSIOK

In another urodele amphibian, Taricha torosa, it has recently been reported that HPL inhibits primary metamorphosis (Cohen et al., 1972). This effect is similar to the manner in which prolactin inhibits metamorphosis in frog tadpoles (Bern et al., 1967; Etkin and Gona, 1967; Gona, 1967, 1968) and in the salamander Ambysto,ma tigrinum (Gona and Etkin, 1970). The Ijrcsent results demonstrate that HPL has a clearcut prolactin-like effect in that it induces second metamorphosis in red efts. However certain unexpected differences emerge between HPL and prolactin in their action in t’he red eft. At, the lo-pg dose level, HPL brought about migration of 9 of the 10 animals into water (90% water drive). In all our previous work we have never been able to induce more than 60% water drive with 10 ng closes of prolactin (Gona et ~2.. 1970, and unpublished data). The HPL appears, therefore, to be even more effective than prolactin in inducing water drive at the lo-pg level. However, at lOO-pg level HPL induced little water-drive effect. Tn a pre-

PLACENTAL

LACTOGEN

TABLE EFFECTS

Group No. 1. 2. 3. 4.

OF HUMAN THYROXINE

~MXNTAL

(Tb)

ACTION

IN

1

LACTOGEN (HPL) ON TAIL HEIGHT IN

Treatment

IV

100 /~g HPL 10 pg HPL 10 fig HPL + 0.01 pg T, Saline

10 10 10 10

liminary study we had found that even 40pg doses of HPL showed a similar failure to effect water drive. This is in clear contrast to the action of prolactin. Large doses of prolactin have never been reported to exhibit such a reversal of the usual stimulation of water drive. In fact we have routinely used 40-100 ,ug doses of prolactin in red efts when we wished to induce second metamorphosis. We have previously reported that small doses (0.1 pg or less) of T,, unlike larger doses, act synergistically with prolactin in the red eft (Gona et al., 1970, 1972). The present results do not indicate synergistic interaction between HPL and T,,. In fact there seems to be an antagonism. Whereas 10 pg HPL alone induced 90% water drive, simultaneous treatment with 0.01 pg T, induced only 50% water drive. Furthermore, there was no detertahle enhancement in the integumentary changes when HPL was supplemented with T, as had hten the case with prolactin. Interestingly, the measurements of tail height provide corroborating evidence for the inbihition of second metamorphosis by either simultaneous treatment with T, or increase in the dose of HPL. The animals treated with 10 pg of HPL alone showed a significant increase in the tail height. On the other hand, animals treated with 10 ,ag of HPL and 0.01 yg of T, as well as those treated with 100 /*g of HPL alone failed to show this effect. Although confirming the prolactinlike action of HPL, the present work clearly demonstrates differences and raises the cplestion of the source of such differences. It is possible that the HPL preparation had an appreciahlc level of contamination

379

NEWTS

WITH .~NU RED EFTS

WITHOUT

Tail height (mm) (mean & SD) 1.37 1.66 1.48 1.38

+ + * *

0.09 0.06 0.05 0.08

P _____ NR <0.005 NS NS

of thyroid hormone or thyrotropin. At the low dose of HPL, any such contamination would be either negligible or synergistic. With the larger dose the amount of impurity may have reached a distinctly inhibitory level. Additive effects of the impurity in the HPL could have been responsible also for the antagonism produced by the rather small (0.01 pg) dose of T,. The present experiment provides no evidence for a synergistic effect of T, on HPL action. Nevertheless, there may be a critical level below which T, is synergistic and above which it is antagonistic with HPL. If such a critical level does exist, it must hc quite different for HPL and for prolactin. HPL has often been considered more prolact.in-like than growth hormone-like. On the ot’her hand, studies of chemical structure of HPL indicate that the homology bet.ween HPL and growth hormone is 96% and is much less hetween HPL and prolactin (Li et al., 1971). On the basis of this evidence, it is not surprising that the action of HPL in the red eft, albeit prolactin-like, is not identical to that of prolactin. If chemical modifications of the prolactin molecule become availahle, it would he of considerable interest to explore their action in the red eft. ACKNOWLEDGMENTS The authors wish to thank Dr. William Etkin for reading the manuscripts and making important suggestions. This work was supported by Grants GB-8737 and GB-12353 from the National Science Foundation. REFERENCES P. chorionic

BECK,

(1972). Lactogenic somatotropin

in

Proc. Sot. Erp. Rid. Med.

activity of human rhesus monkeys. 140, 183-187.

380

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H. A., NrcoLL, C. (1967). Prolactin and Exp. Biol. Med. 126, CHADWICK, C. S. (1940). water drive factor in BERN,

Sot.

Exp.

Biol.

Med.

S., END STAOHMAN, R. C. tadpole growth. Proc. Sot. 518-520. Identity of prolnctin with

l’riturus viridescet~s. 45, 335-337.

Pt,oc.

J. A., LIGHT, P., BERN, H. A., AND ZIPSER, R. D. (1972). Growth and inhibition of metamorphosis in the newt Takha torosa by mammalian hypophysial and placental hormones. Gen. Comp. Bndocriraol. 18, 384-390. ETXIN, W., AND GONA, A. G. (1967). Antagonism between prolactin and thyroid hormone in amphibian development. J. Ezp. Zoo!. 165, 249258. GONA, A. G. (1967). Prolactin as a goilrogenic agent in amphibia. Endocrinology 81, 748-754. GONA, A. G. (lQ68). Radioiodine studies on prolactin action in tadpoles. Gen. Comp. Endocrinol. 11, 278-283. GONA, A. G., AND ETKIN, W. (1970). Inhibition of metamorphosis in Ambystomn tigrinum by prolactin. Gen. Comp. Endocrinol. 14, 589-591. GONA, A. G., PEARLMAN, T., AND ETKIN, W. (1970). Prolactin-interaction in the newt, Diemictylus viridescens. J. Endocrinol. 48, 585590. GONA, A. G., PEARLMAN, T., .~ND GONA. 0. (1973). COHEN,

D. C.,

AND

GREENBERG,

GONA

Effects of prolactin and thyroxine in hypophysectomizcd and thyroidectomized red cfts of the newt Notophthalmus (Diemictylus) kidescens.

Germ.

Comp.

Endooinol.

20,

107-111.

W. C., JR. (1961). Special aspects of the metamorphic processes: stlcond metamorphosis. Amer. Zool. 1, 163-171. GRUMBACH, M. M., KAPLAN, S. I,., SCIARHA, J. J., AND BURR, I. M. (1968). Chorionic growth hormone-pro1act.k (CGP) : Secretion, disposit,ion, biologic activity in man, and postulated function as the LLgrowth hormone” of the second half of pregnancy. Ann. N. Y. Acad. Sci. 148, 501-531. JOSIMOVICH, J. B.. AND MACLEAREN, J. A. (1962). Presence in the human placenta and term serum of a high1.v lactogenic substance immunologically related to pituitary growth hormone. Endnctinology 71, 209-220. LI, C. H., DIXON, J. S.. AND CHUNK, D. (1971). Primary structure of the human chorionic so.mntotropin (HCS) molecule. Science 173, 56 58. SHERWOOD, L. M. (1969). Human placental lactogen: partial analysis of chemical structure and comparison with pituitary growth hormone. In “Progress in Endocrinology” (C. Gual, ed.). pp. 394-401. Excerpta Med. Found.. Amsterdam. GRANT,