Pigment dispersion by prolactin in cultured xanthophores and erythrophores of some fish species

THE JOURNAL OF EXPERIMENTAL ZOOLOGY 275:45-52 (1996)

Pigment Dispersion by Prolactin in Cultured Xanthophores and Erythrophores of Some Fish Species

NORIKO OSHIMA, MIHOKO MAKINO, SHAWICHI IWAMURO, AND HOWARD A. BERN Department of Biomolecular Science, Faculty of Science, Toho University, Miyama, Funabashi, Chiba 274 (N. O., M.M.), and Department of Biology, School of Education, Waseda University, Tokyo 169 (S.I,j, Japan; Department of Integrative Biology and Cancer Research Laboratory, University of California, Berkeley, California 94720 (H.A. B. j

ABSTRACT The direct effects of a pair of tilapia prolactins (tPRL177 and tPRLIS8) and of ovine prolactin (oPRL) on cultured xanthophores and erythrophores isolated from some fish species were examined. Cultured xanthophores of the Nile tilapia, paradise goby, and medaka responded to tPRL177 and oPRL at concentrations of less than 100 nM by pigment dispersion, whereas tPRL188 had an effect only at concentrations of 10 pM or more. Swordtail erythrophores also responded to prolactin even in winter. It is likely, therefore, that in fish species possessing many xanthophores or erythrophores in their skin throughout the year, these cells respond to prolactin all the time. In fall and/or winter, prolactin had little or no effect on xanthophores isolated from the dark chub and rose bitterling or on erythrophores from the tilapia and paradise goby. However, it was shown that xanthophores of the rose bitterling and erythrophores of the tilapia responded better to prolactin in spring and/or summer as compared with fall and/or winter. Such seasonal change in the responsiveness of the cells to prolactin implies the involvement of the hormone in nuptial coloration and an augmentation of red and/or yellow skin color by pigment dispersion in the spawning season. Prolactin-induced pigment dispersion within erythrophores isolated from the red region of tail fins of the rose bitterling assuming nuptial coloration also lends support to this supposition. 0 1996 Wiley-Liss, Inc.

Many important functions of pituitary hormone prolactin have been reported for various teleost fish species. Recently, using split-tail fin prepara- tions, we further demonstrated that the smaller prolactin of the tilapia Oreochromis mossambicus, tPRL177, which consists of 177 amino acids, caused pigment dispersion in xanthophores and erythrophores of the tilapia Oreochromis niloticus, whereas the larger prolactin, consisting of 188 amino acids, tPRL188, was less effective than tPRL177. In addition, ovine prolactin (oPRL) was found to have the same effect (Kitta et al., ’93).

In many teleost fish species, a-melanophore- stimulating hormone (a-MSH) and melanin-con- centrating hormone (MCH) are considered as skin-darkening and -blanching peptides, respectively (Fujii and Oshima, ’86, ’94). Both hormones directly affect melanophores, xanthophores, and erythrophores. However, prolactin molecules did not have a direct effect on melanophores of the tilapia; melanophores in primary culture did not 0 1996 WILEY-LISS, INC.

respond to tPRL177, tPRL188, or oPRL (Kitta et al., ’93). Thus, the action of prolactin seemed to be restricted to xanthophores and erythrophores (“brightly pigmented cells”).

In the present study, we examined the responses to tPRLs and oPRL of cultured pigment cells isolated from several fish species to determine whether the pigment-dispersing effect of prolactin on brightly pigmented cells and the inefficacy of the hormone on melanophores are general prop- erties in teleosts. The role of prolactin in the change in body color of teleosts is also discussed.

MATERIALS AND METHODS The tilapia 0. niloticus, 8-13 cm in total length;

the paradise goby Rhinogobius giurinus, 5-6 cm; the medaka (orange-red variety) Oryzias latipes,

Received September 26, 1995; revision accepted February 26, 1996. Address reprint requests to Prof. Noriko Oshima, Department of

Biomolecular Science, Faculty of Science, Toho University, Miyama, Funabashi, Chiba 274, Japan.

Fig. 1. Photomicrographs showing responses of cultured xanthophores of tilapia to ovine prolactin (oPRL) (B-D) and tilapia prolactin (tPRL)177 (F-H). A Xanthophores equilibrated in physiological saline. B: Fifteen minutes after addition of 100 pM oPRL. C: Fifteen minutes after addition of 10 nM oPRL. D: Fifteen minutes after addition of 100 nM oPRL. E: Thirty minutes afber the beginning of saline rinse. F: Fif- teen minutes after addition of 100 pM tPRL177. G: Fifteen minutes after addition of 10 nM tPRL177. H Fifteen minutes

after addition of 100 nM tPRL177. After each concentration was tested, the cells were fully rinsed with saline for 30 min to attain the initial states shown in A and E. Bar: 50 pn.

Fig. 3. Photomicrographs showing the effect of 10 pM tilapia prolactin (tPRL)lss on cultured xanthophores of tilapia. A: Xanthophore equilibrated in saline. B: Five minutes after addition of tPRLle8. C: Fifteen minutes. D: Thirty minutes. Bar: 50 pm.

PROLACTIN EFFECT ON CULTURED PIGMENT CELLS 41

K - , I 1 I L L I 1 0 .- 0.1 1 10 100 0.1 1 10 100 2

60 - - -+oPRL

-+tPRLies

0.1 1 10 100 0.1 1 10 100 Concentration (nM)

Fig. 2. Relationships of the concentration of tilapia prolactins (tPRLs) and ovine prolactin (oPRL) to the magnitude of pigment-dispersing responses of brightly pigmented cells. Cells were exposed to hormones at various concentrations for 15 min. Full dispersion (100%) was obtained by final applica- tion of 10 nM a-MSH for 15 min. A: Tilapia xanthophores. B: Xanthophores of paradise goby. C: Swordtail erythrophores. D: Tilapia erythrophores isolated from tail fin in January. Each point represents the mean * SE of three (A, B, and C) or four (D) measurements on different animals.

2-3 cm; the dark chub Zacco temmincki, 7-8 cm; the rose bitterling Rhodeus ocellatus ocellatus, 6- 7 cm, and the swordtail Xiphophorus helleri, 5-6 cm, were used. Tilapia with total lengths of 7-8 cm were obtained from a local fish farm near Lake Teganuma (Chiba Prefecture, Japan) in spring and reared in freshwater aquariums all year round. The other fish species were purchased from local commercial sources in fall and maintained in aquariums for 3-8 months. The present materials possess xanthophores and/or erythrophores in their integument. Many xanthophores are always observed in the skin tissue of the tilapia and paradise goby, in addition to melanophores, whereas only a few erythrophores are present in the dermis. In the medaka (orange-red variety) and swordtail, xanthophores and erythrophores are major pigment cells, respectively. Xanthophores are also distributed in the skin of the dark chub and rose bitterling, although their motile activity

is low. Characteristic nuptial coloration appears in the spawning season in the tilapia, dark chub, and rose bitterling. Erythrophores are mainly responsible for the coloration.

Fish were maintained for about 12 hr in deion- ized water containing penicillin-G (100 U/ml, po- tassium salt; Meiji Seika, Tokyo, Japan) and streptomycin sulfate (500 pg/ml, Meiji Seika), then immersed in ice-cold distilled water for anesthe- tization. After the fish was sterilized with 70% ethanol for 15 sec, it was rinsed with Ca", M e - free, Dulbecco's phosphate-buffered saline (CMF- PBS: NaCl 136.9 mM, KC1 2.7 mM, Na2HP04 8.1 mM, KH,PO, 1.5 mM). Caudal and dorsal fins were excised and cut into pieces about 2 mm2 in CMF-PBS. The skin pieces and scales from the dorsum were put into an Erlenmeyer flask filled with CMF-PBS and washed five times with saline. They were treated with CMF-PBS containing 0.02% ethylenediamine-tetraacetic acid (EDTA-3Na 3Hz0; Dojindo Laboratory, Kumamoto, Japan) for 10-15 min, then with 0.25% collagenase (type 11; Sigma Chemical, St. Louis, MO) solution made up in CMF-PBS for 30 min. The suspension of dissociated cells was transferred to a centrifuge tube. The cells collected by centrifugation at 150 g for 3 min were then resuspended in the culture medium, which consisted of a solution containing 9.4 g/l powdered Eagle's minimal essential medium (Nissui Seiyaku, Tokyo, Japan); 100,000 UA penicillin-G (Meiji Seika); 100 mg/l streptomycin sulfate (Meiji Seika); 300 mgfl L-glutamine powder (Wako Pure Chemical, Osaka, Japan); and 1,280 mgA NaHC03 (Wako Pure Chemical), double-distilled water, and fetal bovine serum (GIBCO Labo- ratories, Grand Island, NY). They were mixed in a volume ratio of 8:l:l (v/v). In order to isolate more pigment cells, the supernatant collagenase- rich solution was again decanted into the flask. Treatment of scales and fin pieces with collagenase was repeated five times. Isolated pigment cells were placed on coverslips in sterilized dis- posable plastic dishes and cultured for 24-48 hr at 25°C in a water-jacketed C02 incubator.

The larger and smaller prolactins of the tilapia, which consist of 188 and 177 amino acids, respectively, were isolated from the pituitary of 0. mossambicus as described by Yamaguchi et al. (1988). oPRL, purchased from Sigma, was purified further on a reverse-phase high-performance liquid chromatography (HPLC) column, TSK TMS250T (4.6 x 75 mm; Tosoh, Tokyo, Japan) using a linear gradient of 1 0 4 0 % acetonitrile (MeCN) containing 0.1% trifluoroacetic acid for

48 N. OSHIMA ET AL.

Fig. 4. Photomicrographs showing responses of cultured xanthophores of paradise goby to tilapia prolactin (tPRL)177 (B-E) and ovine prolactin (oPRL) (G-J). A: Xanthophore equilibrated in saline. B: Fifteen minutes after addition of 100 pM tPRL177. C: Fifteen minutes after addition of 1 nM. D: Fifteen minutes after addition of 10 nM. E: Fifteen minutes after addition of 100 nM. F: Other xanthophores equilibrated in saline. G: Fifteen minutes after addition of 100 pM oPRL. H Fifteen minutes after addition of 1 nM. I: Fifteen minutes after addition of 10 nM. J: Fifteen minutes after addition of 100 nM. After each concentration was examined,

cells were rinsed with saline for 30 min to attain the initial states shown in A and F. Bar: 50 pm.

Fig. 5. Photomicrographs showing responses of medaka xanthophores in culture t o tilapia prolactins (tPRLs) at 100 nM. tPRL188 (100 nM) did not cause pigment dispersion within 30 min. A Xanthophore equilibrated in saline. B: Fifteen minutes after addition of tPRL177 (100 nM). C: Thirty minutes. D: Another xanthophore equilibrated in saline. E: Thirty minutes after addition of 100 nM tPRLl88. Bar: 50 pm.

PROLACTIN EFFECT ON CULTZTRED PIGMENT CELLS

TABLE 1. Responses of fish xanthophores to prolactins and a-MSH

49

Species

Nile tilapia

Paradise goby

Medaka

Dark chub

Rose bitterling

(Oreochromis niloticus)

(Rhinogobius giurinus)

(Oryzias latipes)

(Zacco temmincki)

(Rhodeus ocellatus ocellatus)

a-MSH (10 nM)

Time of experiments

March-Nov.

Oet .-Nov.

Nov.

Nov.-Dec.

Nov.-Dec. June-Julv

more than 25 min at a flow rate of 1.0 mumin. Absorbance at 220 nm was monitored with an L- 3000 spectrophotometer (Hitachi, Tokyo, Japan). The main peak eluting at 52% MeCN was collected and used as the test substance. a-MSH (Sigma) at 10 nM was applied for 15 min to ob- tain maximal dispersion of pigment granules.

A coverslip with cultured pigment cells attached was mounted on a perfusion chamber filled with physiological saline solution (NaC1 125.3 mM, KC1 2.7 mM, CaClz 1.8 mM, MgClz 1.8 mM, D-glucose 5.6 mM, Tris-HC1 buffer 5.0 mM; pH 7.21, and the assembly was transferred onto the stage of a light microscope. After the cells were fully washed with saline, responses of pigment cells to prolactins and a-MSH were recorded with a real-time image ana- lyzer (RUZEX F, Nireco, Tokyo, Japan; cf. Oshima et al., '88). In some experiments, cellular responses were continuously microphotographed and ana- lyzed with RUZEX F. All experiments were per- formed at room temperature (20-26OC).

RESULTS Many xanthophores in primary cell culture

maintained the pigment-aggregated state in physiological saline solution. Cultured xanthophores isolated from tilapia skin responded to tPRL177 (the smaller prolactin of 0. mossambicus) and oPRL by pigment dispersion in a dose-dependent manner (Figs. 1, 2A). At 100 pM, the extent of pigment dispersion caused by tPRL177 was signifi- cantly higher than that by oPRL (Student's t test, P < O.Ol) , but there were no significant differences in dispersion levels between tPRL177- and oPRL- treated xanthophores at any other concentrations. Although tPRLls8 (the larger tilapia prolactin) at 100 nM failed in the induction of pigment dispersion, it caused dispersion at 10 pM (Fig. 3).

Responses to prolactins of xanthophores isolated

from the paradise goby were fundamentally similar to those of the tilapia xanthophores. There was no significant difference in effectiveness between tPRL177 and oPRL (Figs. 2B, 4). In cultured xanthophores of the medaka, tPRL177 at 100 nM also induced pigment dispersion but tPRLlB8 at the same concentration did not, as shown in Figure 5. When tPRLlss was applied at 10 pM, pigment dispersion within medaka xanthophores was observed.

From these results, it was concluded that tPRL177 and oPRL were effective in inducing pigment dispersion in xanthophores of some fish species.

By contrast, xanthophores from the dark chub and rose bitterling did not respond to prolactin in fall and winter; however, complete dispersion did occur in response to a-MSH (10 nM), as was the case for xanthophores of all fish species tested. In summer (June-July), the responsiveness to oPRL of xanthophores from the rose bitterling assuming nuptial coloration was tested. Surprisingly, the pigment cells responded well to oPRL by pigment dispersion. The results are summarized in Table 1.

Next, responses of erythrophores to prolactin were examined. In physiological saline, pigment granules within swordtail erythrophores in primary culture were aggregated. The cells responded to tPRL177 and oPRL by dose-dependent dispersion of pigment. There was no significant difference in effectiveness between the two hormones (Fig. 2C). tPRL188 did not affect swordtail erythrophores at 100 nM, but it caused appre- ciable pigment dispersion at 10 pM.

In the Nile tilapia, a few erythrophores are present, only in the fins usually, whereas many melanophores and xanthophores are seen in the scales and fins. With a few exceptions, cultured erythrophores from tilapia retained their pigment in the aggregated state, and in such cells, tPRL177 and oPRL caused only a low level of pigment dis-

50 N. OSHIMA ET AL.

Fig. 6. Photomicrographs showing responses to 100 nM ovine prolactin (oPRL) of a cultured erythrophore isolated from a fin of tilapia manifesting nuptial coloration in May. A: Erythrophore equilibrated in saline. B: One and a half min- Utes after addition of 100 nM oPRL. C: Three minutes. D Five minutes. Bar: 50 pm.

Fig. 7. Photomicrographs showing responses to ovine pro-

lactin (oPRL) and tilapia prolactin (tPRL)177 at 100 nM of a cultured erythrophore isolated from a fin of rose bitterling manifesting nuptial coloration in July. A: Erythrophore equilibrated in saline. B: Five minutes after addition of 100 nM oPRL. C: Fifteen minutes. D Thirty minutes after the beginning of saline rinse. E: Fifteen minutes after addition of 100 nM tPRL177. F Thirty minutes. Bar: 50 pm.

persion (Fig. 2D). For example, the extent of pigment dispersion induced by treatment with tPRL177 at 100 nM for 15 min was about 8.7% of the maxi- mum response caused by a-MSH (10 nM). tPRLIa8 had no effect even at 10 pM. Through spring into summer, however, the number of erythrophores in the fins increases in some tilapias, resulting in

augmentation of the reddish tinge. Some tilapias (male) clearly manifest nuptial coloration: the peripheral region of the dorsal and tail fins turns red. As shown in Figure 6, erythrophores isolated from such individuals responded very well to oPRL, which was used in place of fish prolactins owing to its greater availability.

PROLACTIN EFFECT ON CULTURED PIGMENT CELLS

TABLE 2. Responses of fish erythrophores to prolactins and a-MSH

51

tPRLi77 tPRL188 oPRL a-MSH Time of (100 nM) (100 nM) (10 &M) (100 nM) (10 nM1 experiments

Nile tilapia - - - - + Jan. + + April-June

Paradise goby - - - - + Feb.-March Swordtail + - + + + Feb.

Rose bitterling + - + + + June-July (Xiphophorus helleri)

Characteristic nuptial coloration is also seen in male rose bitterlings in the spawning season. Many erythrophores appear in the middle region of the tail fin and in the peripheral region of the dorsal and anal fins, whereas few erythrophores are present in other seasons. When cultured erythrophores isolated from the reddish region of the tail fins were treated with tPRLIy7 and oPRL at 100 nM, pigment dispersion was observed, as shown in Figure 7, whereas tPRLL8* at the same concentration did not have this effect. tPRLla8 af- fected the cells at 10 pM.

In the paradise goby, erythrophores did not respond t o tPRLs and oPRL from February to March. Results on erythrophore responses are summarized in Table 2.

Prolactin had no effect on cultured melanophores in any teleost species examined in the present study.

DISCUSSION The direct action of tPRL177 and oPRL on xan-

thophores and erythrophores was further demonstrated in several fish species in addition to the Nile tilapia. In xanthophores and erythrophores responsive to tPRL177, oPRL was also effective in inducing pigment dispersion. In teleost fish possessing many xanthophores or erythrophores in their skin throughout the year, response to prolactin appears to occur all year round. For example, xanthophores of high density are always observable in scales of the Nile tilapia, medaka, and paradise goby. In the swordtail, erythrophores are the major pigment cells responsible for skin color. These cells respond to prolactin throughout the year.

However, neither peptide had an effect on brightly pigmented cells from late autumn to early spring in some species. Therefore, in these latter species, responsiveness to prolactin may be asso- ciated with breeding season. The results obtained on tilapia erythrophores clearly suggest seasonal changes in the sensitivity of the brightly pig-

mented cells to prolactin: erythrophores isolated from tilapia in January showed only slight dispersion in response to tPRL177 and oPRL, but erythrophores isolated from individuals with reddish tail fins from April to June responded well to oPRL by pigment dispersion. The effect of tPRL177 has not yet been examined. Erythrophores dissociated from the red region of fins of rose bitterlings manifesting nuptial coloration also responded to tPRL177 and oPRL. We are currently examining in detail seasonal changes in the responsiveness to prolactin of xanthophores and erythrophores of certain fish species, but the results imply that prolactin functions not only to disperse pigment in brightly pigmented cells but also to increase the number of erythrophores, resulting in nuptial coloration, a kind of “morphological color change.” The role of a-MSH in the background adaptation of lower vertebrates may be a similar case. When animals are adapted to a black background, a-MSH induces pigment dispersion within melanophores, leading to skin darkening (“physiological color change”). Plasma levels of MSH in animals increase during adaptation to a black background (Jenks et al., ’77; Wilson and Morgan, ’79). Morphological color change, the increase in the number and size of melanophores as well as in the amount of melanin, may follow the MSH-induced physiological color change and is also controlled by a-MSH (Bagnara and Hadley, ’73; van Eys and Peters, ’81).

In newts, nuptial coloration of the tail was induced by injection of prolactin and androgen, although the coloration was not due to an increase in the amount of pigment but rather to morphological changes in the dermis: in the tail skin with coloration, iridophore processes elongate beneath the basal lamina, while melanophore processes extend above iridophores in the skin without coloration (Negishi et al., ’94). In teleost fish, too, prolactin might take part in the increase in erythrophores in cooperation with other hormones, such as androgen, leading to nuptial coloration.

52 N. OSHIMA ET AL.

There is an important difference between prolactin and a-MSH: prolactin causes pigment dispersion only in brightly pigmented cells, whereas MSH affects all kinds of chromatophores, resulting in skin darkening i n many cases. Dispersion of pigment by prolactin only in xanthophores and erythrophores can produce yellowish or reddish color of the skin, which could be involved in com- munication with conspecifics.

The ineffectiveness of tPRL188 compared with tPRL177 is noteworthy. The former may have no actual effect on brightby pigmented cells in vivo because this peptide did not induce pigment dispersion even at 100 nM. The degree of similarity between the amino acid sequences of tPRLs (69%, 130 identical residues; Yamaguchi et al., '88) is low compared with that between pairs of other teleostean PRLs: a pair of chum salmon PRLs and of common carp PRLs differ from each other by only four residues and one residue, respectively (Yasuda et al., '86, '87). The similarity of tPRLlss to the other teleostean PRLs is greater than that of tPRL177. That is, tPRLls8 is about 69% identical to salmon PRL and 64% identical to carp PRL, while tPRL,,, is about 56% identical to salmon PRL and 51% identical to carp PRL. Although both tPRLs fulfilled osmoregulatory functions (Specker et al., '85a), Yamaguchi et al. ('88) speculated that conservative evolutionaiy pressure on tPRLs re- mained on tPRLls8, leaving tPRLlT7 free to change and take on other functions. Further, Specker et al. ('85b) reported that tPRL177 did not promote an increase in the length and weight of intact ju- venile tilapia. Therefore, we now propose that tPRL177 is a renegade I'RL molecule which has lost growth hormone-like characteristics and is actively involved in a change in skin color, affect- ing xanthophores and erythrophores.

ACKN0WL:EDGMENTS We are grateful to Drs. R.S. Nishioka, D.S. King,

E.G. Grau, N.H. Richman, and J.L. Specker and their associates for their aid in the acquisition of purified tilapia hormones. This work was sup- ported by MESCJ grant 07640915 (N.O.) and by

LITERATURE CITED Bagnara, J.T., and M.E. Hadley (1973) Chromatophores and

Color Changes. Prentice-Hall, Englewood Cliffs, NJ. Fujii, R., and N. Oshima (1986) Control of chromatophore

movements in teleost fishes. Zool. Sci., 3:13-47. Fujii, R., and N. Oshima (1994) Factors influencing motile

activities of fish chromatophores. In: Advances in Compara- tive and Environmental Physiology, vol. 20. R. Gilles, ed. Springer-Verlag, Berlin, pp. 1-54.

Jenks, B.G., A.P. van Overbeeke, and B.F. McStay (1977) Syn- thesis, storage and release of MSH in the parts intermedia of the pituitary gland of Xenopus laevis during background adaptation. Can. J. Zool., 55:922-927.

Kitta, K., M. Makino, N. Oshima, and H.A. Bern (1993) Effects of prolactins on the chromatophores of the tilapia, Oreochromis niloticus. Gen. Comp. Endocrinol.,

Negishi, S., T. Yoshida, F. Toyoda, and S. Kikuyama (1994) Fine structure and pigment determination of cynops skin representing nuptial coloration induced by the injection of hormones. Pigment Cell Res., 7:370.

Oshima, N., M. Suzuki, N. Yamaji, and R. Fujii (1988) Pig- ment aggregation is triggered by a n increase in free cal- cium ions within fish chromatophores. Comp. Biochem. Physiol. A, 91:27-32.

Specker, J.L., D.S. King, R.S. Nishioka, K. Shirahata, K. Yamaguchi, and H.A. Bern (1985a) Isolation and partial characterization of a pair of prolactins released in vitro by the pituitary of a cichlid fish, Oreochromis mossambicus. Proc. Natl. Acad. Sci. USA, 82:7490-7494.

Specker, J.L., D.S. King, R.J. Rivas, and B.K. Young (198513) Partial characterization of two prolactins from a cichlid fish. In: Prolactin, Basic and Clinical Correlates, vol. 1. R.M. MacLeod, M.O. Thorner, and U. Scapagnini, eds. Liviana Press, Padua, Italy, pp. 427-436.

van Eys, G.J.J.M., and P.T.W. Peters (1981) Evidence for a direct role of a-MSH in morphological background adaptation of the skin in Sarotherodon mossambicus. Cell Tissue Res., 217:361-372.

Wilson, J.F., and M.A. Morgan (1979) a-Melanotropin-like sub- stances in the pituitary and plasma of Xenopus Eaeuis in relation to colour change responses. Gen. Comp. Endocrinol.,

Yamaguchi, K., J.L. Specker, D.S. King, Y. Yokoo, R.S. Nishioka, T. Hirano, and H.A. Bern (1988) Complete amino acid sequences of a pair of fish (tilapia) proladins, tPRL177 and tPRLl88. J. Biol. Chem., 263:9113-9121.

Yasuda, A., H. Itoh, and H. Kawauchi (1986) Primary structure of chum salmon proteins: Occurrence of highly con- served regions. Arch. Biochem. Biophys., 244:528-541.

Yasuda, A., K. Miyazima, H. Kawauchi, R.E. Peter, H.-R. Lin, K. Yamaguchi, and H. Sano (1987) Primary structure of common carp prolactins. Gen. Comp. Endocrinol.,

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Pigment dispersion by prolactin in cultured xanthophores and erythrophores of some fish species