Effect of Sex on the Development of Melanoma in Hybrid Fish of the Genus Xiphophorus'

[CANCER RESEARCH 31, 725â€”729,June 19711 Effect of Sex on the Development of Melanoma in Hybrid Fish of the Genus Xiphophorus'

Michael J. Siciliano,2 Alfred Perlmutter, and Edward Clark Biology fM. J. S.! and Physics fE. C.J Departments, Zeckendorf Campus, Long Island University, Brooklyn, New York 11201, and Biology Department, Washington Square Campus, New York University, New York, New York 10003 fA. P.1

SUMMARY from the platyfish parent that controls the macromelanophores producing a spotting pattern. In F1 The effect of sex on the development of melanoma in F1 platy-swordtail hybrids receiving the spotting gene, the fish produced by reciprocal artificial inseminations between macromelanophores behave abnormally, producing the state of the platyfish, Xiphophorus maculatus (Guenther) from the atypical melanosis and, in some cases, melanoma, a neoplastic Rio Jamapa (strain 163A), and the swordtail, Xiphophorus state in which abnormal growth of pigment cells is invasive and helleri strigatus (Regan) from the Rio Sarabia (pedigree 1678), destructive to other tissues and forms nodular lesions. was studied. Macromelanophore-stimulating factors from the swordtail By 300 days after birth, all F1 fish were sexually mature have a significant function in induction of melanosis and with a significantly higher percentage ofmales produced from melanoma. When F1 melanotic hybrids are backcrossed to the crosses where the female parents were platyfish than from swordtails, the degree of melanosis in these backcross hybrids the reciprocal crosses. A significantly higher frequency of is usually greater than and never less than that in the original melanoma was observed in mature F1 , spotted dorsal, male hybrids, whereas backcrosses to platyfish produce a lesser fish than in females, regardless of the cross. degree of melanosis than that in the original melanotic The rate of melanotic development was faster in broods of hybrids. Thus, the development of melanoma in hybrid fish is the cross that eventually contained mostly male fish. This considered to be the result of genic imbalance. difference became apparent at a time which corresponded to Haussler (16) suggested that the male sex hormone was the initiation of testicular and androgen function. involved in the process of melanotic development since only It therefore was concluded that the development of male melanotic hybrids eventually developed tumors. Kosswig melanoma in fish hybrids is enhanced in males and that this (18) challenged this hypothesis since he had found tumors in effect probably reflects an augmentation of the melanotic female as well as male hybrids. The latter view was supported process by the male sex hormones. The similarity of this effect by Berg and Gordon (8), who stated that the sex hormones in certain mammalian neoplasms with a genetic basis was were unrelated to melanosis. However, Anders (3) believed discussed. that steroid hormones did affect pigmentation. Atz (7) reported that atypical melanosis (although not melanoma) appeared to be more pronounced in male than in female INTRODUCTION hybrids. He stated that no connection between sex and melanosis had been previously established experimentally in Breeding experiments between the swordtail, Xiphophorus platy-swordtail hybrids. He suggested that the question was helleri (Heckel), and the platyfish, Xiphophorus maculatus still unresolved and needed further study since his observations (Guenther), poeciliid fish indigenous to the river systems were made on only 2 1 fish, of unknown age and of several draining Eastern Central America, have been performed over different cross-types (F1 , F2 , and backcross). the last 50 years. Results of experiments from laboratories in the United States and in Germany were reviewed by Gordon (1 1) and Anders (3). MATERIALS AND METHODS It was discovered that, when certain platyfish were crossed with swordtails, many of the F1 hybrid fish were atypically All fish used in the present experiments were the melanotic, i.e., pigmentation extended beyond the normal descendants of fish obtained from the Fish Genetics limits of spotting pattern present in the platyfish parent. One Laboratory of the New York Zoological Society in 1963. The of the prerequisites for this condition is the presence of a gene swordtails were descendants of Xiphophorus helleri strigatus (Regan) collected from the Rio Sarabia, pedigree 1678. The I Portion of a thesis submitted by M. J. S. in partial fulfillment of the platyfish were descendants of X. maculatus (Guenther) requirements for the degree of Doctor of Philosophy, New York collected from the Rio Jamapa, strain 163A. The latter have University at Washington Square in New York City. been inbred for at least 33 generations and are distinguished by 2 Present address: Section of Medical Genetics, Department of Biology, The University of Texas M.D. Anderson Hospital and Tumor a macromelanophore pattern gene, spotted dorsal (Sd). This Institute at Houston, Houston, Texas 77025. gene is dominantly inherited, causing appearance of a dark Received November 30, 1970; accepted January 25, 1971. spot on the dorsal fin in both male and female platyfish.

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Table 1 Frequency of melanomain spotted dorsal F, malesand females at approximately 300 daysafter birth

Male FemaleLevel

TotalBroodtumorous ofNo. Total No. (p)3A. no. tumorous no.significance

Female swordtail X male platy 153b2 0 0 8 651 2 5 6Sb3 1 3 1473 4 9 1013b3 3 6 11326 3 7 1132b8 10 3 22Subtotal 10 8

95% A26 33 49 Melanoma78.851.6<0.011B.

Female platy x male swordtail 448 5 7 2 020 8 0 162 0 1 01525 2 0 1615b20 27 4 1515c4 25 9 1215d0 4 8 10532 0 8 1Subtotal 2 1

59% B66 75 33 55.9<0.01Total92Melanoma88.0

154% 108 82 Melanoma85.2 53.2<0.01

Reciprocal matings (female swordtails X male platyfish, and 100

female platyfish X male swordtails) were accomplished via U) modification of artificial insemination described by Clark (9). z CO The inseminated female fish were kept in 5-gallon aquaria 80 @.1 filled with conditioned tap water containing an abundance of CO Ui from female swordtail the filamentous alga, Nitella. After birth of a brood (primary 60 brood), the parent was immediately removed to another tank to await the birth of subsequent broods. Young fish were raised and maintained in 5. and 10-gallon x 40 aquaria with conditioned water under constant aeration; the water was changed about once a month. Water temperature CO @- Ui was maintained at 24 Â±3Â°.Fish were fed a varied diet Ui 0. 20 @ Ui consisting of ground, cooked bovine liver; dry, prepared Ui commercial food; microworms; cooked egg yolk; and brine 0. shrimp nauplii. Thirty days after birth, the fish of a brood were redistributed so that there were no more than 12 in 5 io 100 140 gallons of water. NUMBER OF DAYS AFTER BIRTH From 10 to 300 days after birth, spotted dorsal F1 fish Chart 1. The percentage of spotted dorsal (Sd), F, hybrids from were removed from their tank about every 20 days and placed female platyfIsh and from female swordtails, expressing atypical in a l.gallon plastic aquarium. Each fish was examined and melanosis, from 20 to 160 days after birth. scored with respect to the expression of the spotted dorsal trait. They were scored into 1 of 3 categories: (a) spotted destruction in addition to black spotting on the dorsal fin and dorsal (black spotting in the dorsal fin); (b) atypical melanosis adjacent regions). (black spotting on the caudal peduncle and/or body of the fish About 300 days after birth, the sex of each F1 fish was in addition to the dorsal fin); (c) tumor or melanoma determined. Ability to recover sperm from a fish indicated a (appearance of black nodular lesions and/or normal tissue male; eggs seen through the body wall indicated a female.

726 CANCER RESEARCH VOL.31

When sex was in doubt by these methods, the fish was 80

sacrificed and the gonad was examined. @0 V ., The F1 fish receiving the Sd gene of the reciprical crosses C,) F1 from female ploty â€œ were compared with respect to sex ratio, rate of atypical F- melanosis and melanoma development, percentage of adult h60 I 1 males with melanoma, and percentage of adult females with I melanoma. The percentages of adult, spotted dorsal males with @ melanoma were also compared with the percentages of 40 Ia. U) melanoma in adult, spotted dorsal females both within Oco /1 Li reciprocal crosses and in the pooled data. @ Differences between these comparisons were tested for U. statistical significance by making use of the binomial I-z 20 Li probability distribution.3 Individual broods were tested for 0 F1 from female swordtoil Li the significance of their variance from the results of the overall 0. experiment. All calculations were done on the IBM-i 130 @ computer system. @Oâ€˜60 â€˜ 00 â€˜ 40 â€˜ 80 â€˜220 260 300 NUMBEROF DAYS AFTER BIRTH

RESULTS Chart 2. The percentage of spotted dorsal (Sd), F, hybrids from female platyfIsh and from female swordtail expressing melanoma from Seventeen broods, initially totaling 335 spotted dorsal F1 20 to 300 days after birth. fish, were produced. Eight broods of 164 fish resulted from crosses between female swordtails and male platyfish, and 9 Beyond 120 days after birth, practically all fish from both broods of 17 1 fish were produced from reciprocal crosses crosses were atypically melanotic (Chart 1). between female platyfish and male swordtails. At 300 days From 100 days after birth through the entire experimental after birth, 128 (78%) of the 164 fish produced from the period, a significantly higher percentage of F1 fish from the female swordtail and male platyfish crosses were still living, as crosses that produced mostly male fish were tumorous than were 134 (78%) of the I 7 1 fish from the reciprocal crosses. those from the reciprocal cross (Chart 2). Losses in both groups could be attributed to handling. The difference between the percentages of males with melanoma (79% versus 88%) in the reciprocal crosses (Table 1) DISCUSSION was not significant nor was it for females (52% versus 56%). At The development of a significantly higher percentage of approximately 300 days after birth, 92 (85%) of 108 males melanoma, by 300 days after birth in male as compared to had melanoma, whereas 82 (53%) of 154 females were female, spotted dorsal, F1 hybrids, has been clearly indicated tumorous. This difference is statistically highly significant (p < 0.01). The statistical analysis showed further that in none in the present study. This was true whether the hybrids were produced from either the female platyfish or female swordtail. of the broods in either sex did percentage of tumorous fish The higher incidence of atypical melanosis from 60 to 120 vary significantly from the percentage of tumorous fish days after birth and melanoma after 100 days after birth in F1 calculated for that sex from the pooled data. fish from female platyfish as opposed to F1 fish from female The tendency of a higher percentage of males to develop swordtails is considered to be a reflection of the higher melanoma existed in F1 fish from the female swordtail and percentage of males produced by the former cross. This male platyfish breeding, as well as those from the reciprocal interpretation is considered valid since there was no crosses. In the former, 79% of the 33 males had tumors significant difference at 300 days after birth between the whereas only 52% of the 95 females had tumors (p < 0.01). In percentages of males with melanoma or between the the female platyfish and male swordtail hybrids, 88% of 75 percentages of females with melanoma when the reciprocal males had tumors as opposed to 56% of 59 females (p < 0.01). crosses were compared. The basis of the different sex ratios in A significantly higher percentage of male fish (75 of 134 or the reciprocal crosses is the subject of another study which 56%) were produced from the female platyfish and male will be reported later. swordtail crosses than from the reciprocal crosses (33 of 128 The value of tracing the developmental course of atypical or 26%). At 300 days after birth, all fish were sexually well melanosis among members of the reciprocal crosses from birth defined and functional, i.e., males produced sperm and females to 300 days after birth is indicated. Clearly, if only mature fish produced eggs. had been examined and compared, differences in the rate of From 60 to 120 days after birth, a significantly higher melanotic development would not have been observed, since percentage of F1 hybrids from the crosses which produced beyond 120 days practically all F1 fish were atypically mostly male fish (female platyfish X male swordtail) were melanotic. atypically melanotic than those from the reciprocal crosses. The fact that the higher frequency of atypical melanosis was observable in broods containing mostly male fish and that this 3 Binomial probability distribution is applicable to samples of any size from populations in which objects occur in 2 categories, e.g., difference was limited to the period from 60 to 120 days after melanoma or no melanoma, male or female, etc. (20). birth is important. This period corresponds to the time when

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1971 American Association for Cancer Research. Michael J. Siciliano, Alfred Perlmutter, and Edward Clark the anal fin begins to develop thickened marginal fin rays ACKNOWLEDGMENTS (indicative of gonopodial development in males). At birth, there is no sexual dimorphism in the gonad of fish of this We thank the following individuals at Long Island University : Dr. genus (2 1). Since gonopodial development has been linked to Anthony lovino and Mr. Joseph Bottemidi for their aid in procuring testicular development (12, 14) and directly to the male sex space, equipment, and supplies; Mr. Mark Taddonio, Mr. Harold Kucine, and Mr. Sergio Abreu for their assistance in the care and hormones (13, 14, 22), it is suggested that the higher incidence feeding of the fish population; and Mr. Anthony Brogna for making the of atypical melanosis in mostly male broods and the resultant computer facilities available. We owe special thanks to Dr. Klaus higher incidence of melanoma in adult males is a manifestation Kailman of the Osborn Laboratories of Marine Sciences for providing us of testicular function and elaboration of male sex hormones. It with the inbred strains from which our fish were developed, and to Dr. is proposed, therefore, that male sex hormones enhance the David E. Anderson of The University of Texas M. D. Anderson Hospital tumorigenic process initiated by the genic imbalance in the and Tumor Institute at Houston for his reading of the manuscript. fish hybrid melanoma system. A relationship between sex and melanosis was suggested by Atz (7) although not observed in an exhaustive study by Berg REFERENCES and Gordon (8). Success in establishing this relationship in the present study may have been caused by a fortuitous choice of 1. Agnew, L., and Gardner, W. The Incidence of Spontaneous the strain of swordtail and the ability, through the use of Hepatomas in C3H, C3H (Low MilkFactor) and CBAMiceand the artificial insemination, to produce large numbers ofhybrids of Effect of Estrogen and Androgen on the Occurrence of These both reciprocal cross types and sexes. In agreement with Tumors in C3H Mice. Cancer Res., 12: 757â€”761,1952. present findings, Anders (3), using a wide variety of swordtail 2. Allen, A. C. The Skin, pp. 987â€”991.New York: Grune & Stratton, subspecies and artificial insemination to produce F1 hybrids, Inc., 1967. reported the possibility of a sexual effect on melanosis. 3. Anders, F. Tumor Formation in Platyfish-Swordtail Hybrids as a Kallman (1 7), in a review, referred to the high degree of Problem of Gene Regulation. Experientia, 23: 1â€”10,1967. 3a. Anderson, D. E. Unusual Inheritance of a Genetic Type of sexual abnormalities seen in the past among hybrids of the Cutaneous Melanoma in Man. In: V. Riley (ed.), Pigmentation: Its genus Xiphophorus. For example, Gordon (10) reported Genesis and Control. New York: Appleton-Century-Crofts, in widespread infertility among F1 fish of this hybrid system. In press, 1971. the present investigation, the X. helleri strigatus from the Rio 4. Anderson, D. E., Smith, J. L., and McBride, C. M. Hereditary Sarabia (a strain not used by Gordon, Kosswig, and other early Aspects of Malignant Melanoma. J. Am. Med. Assoc., 200: workers) produced sexually normal F1 fish. The spotted dorsal 741â€”746, 1967. F1 male hybrids produced active, motile sperm; the females 5. Andervont, H. B. The Occurrence of Spontaneous and Induced had masses of yolky eggs; and both produced F2 and Pulmonary and Liver Tumors in Strain .C3H Mice. Public Health backcross offspring. The ability to observe a sexual effect in Rept.,54: 1158â€”1169,1939. the present study was likely the result of the development of 6. Attie, J. N., and Khafif, R. A. Etiology of MelanoticTumors. In: Melanotic Tumors, pp. 160â€”183. Springfield, Ill.: Charles C functional gonads in the experimental animals. Thomas, Publisher, 1964. The high incidence and earlier onset of melanoma in male 7. Atz, J. W. Effects of Hybridization of Pigmentation in Fishes of hybrid fish is in conformity with certain findings on melanoma the Genus Xiphophorus. Zoologica, 47: 153â€”181,1962. in men. Recent reviews of clinical data have shown that men 8. Berg, 0., and Gordon, M. Relationship of Atypical Pigment Cell who have melanoma have a poorer prognosis and a lower Growth to Gonadal Development in Hybrid Fishes.In: M.Gordon survival rate than do women (2, 19). Anderson (3a) has (ed.), Pigment Cell Growth, pp. 43â€”72. New York: Academic indicated the possibility of an earlier onset of familial Press, Inc., 1953. melanoma in men than women. An influence of hormones is 9. Clark, E. A Method for Artificial Insemination in Viviparous also indicated by the unusual behavior of pigmented lesions in Fishes. Science, 112: 722â€”723, 1950. human beings (6) during critical stages of peak endocrine 10. Gordon, M. Effects of Five Primary Genes on the Site of Melanomas in Fishes and the Influence of Two Color Genes on activity (puberty, pregnancy, and menopause). Their Pigmentation. In: Biology of Melanomas, Special Publication Significantly higher frequencies of hepatoma in male mice, of the New York Academy ofSciences, Vol. 4, pp. 216â€”268. New as opposed to female mice, have also been reported (5, 15, York: New York Academy ofSciences, 1948. 23). Androgens were again suggested as the enhancing agents 11. Gordon, M. Physiological Genetics of Fishes. In: M. E. Brown (I). (ed.), The Physiology of Fishes, Vol. 2, pp. 431â€”496. New York: In each of the above examples (human melanoma and Academic Press, Inc., 1957. mouse hepatoma), a genetic etiology has been demonstrated 12. Grobstein, C. Endocrine and Developmental Studies of Gonopod (4, 15). Thereis no questionof the geneticetiology of Differentiation in Certain Poeciliid Fishes. I. The Structure and melanoma in fish hybrids. Since androgens appear to have Development of the Gonopod in Platypoecilus maculatus. Univ. enhanced tumor development in each of these instances, Calif. (Berkeley) Publ. Zool., 47: 1â€”22, 1940. further studies on the effects of androgens on cancers with a 13. Grobstein, C. The Role of Androgen in Declining Regenerative Capacity during Morphogenesis of the Platypoecilus maculatus known or suspected genetic etiology might be valuable. Gonopodium. J. Exptl. Zool., 106: 3 13â€”344,1947. Furthermore, the fish hybrid system should be most useful in 14. Grobstein, C. Optimal Gonopodial Morphogenesis in Platypoecilus elucidating the interrelationships between sexual development, maculatus with Constant Dosageof Methyl Testosterone.J. Exptl. hormone production, genetic constitution, environmental Zool., 109: 215â€”237,1948. influences, and melanogenesis. 15. Hancock, R. L., and Dickie, M. M. Biochemical, Pathological, and

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Genetic Aspects of a Spontaneous Mouse Hepatoma. J. Natl. 20. Sokal, R. R., and Rohif, F. J. Introduction to Probability Cancer Inst., 43: 407â€”415, 1969. Distributions: Binomial and Poisson. In: Biometry, pp. 65â€”98. San 16. Haussler, G. Ueber Malanombildungen bei Bastarden von Francisco: W.H. Freeman and Co., 1969. Xiphophorus helleri und Platypoecilus maculatus.Var Rubra. Kim. 21. Tavolga, W. N. Embryonic Development of the Platyfish, the Wochschr. 7: 1561â€”1562, 1928. Swordtail and Their Hybrids. Bull. Am. Mus. Natl. Hist., 94: 17. Kailman, K. D. Genetics and Geography of Sex Determination in 161â€”229, 1949. the Poeciliid Fish, Xiphophorus maculatus. Zooligica, 50: 22. Turner, C. L. The Rate of Morphogenesis and Regeneration of the 151â€”190,1965. Gonopodium in Normal and Castrate Malesof Gambusiaaffinis. J. 18. Kosswig, C. Ueber Kreuzungen zwischen den Teleostiern Exptl. Zool., 106: 215â€”243,1947. Xiphophorus helleri und Platypoecilus maculatus. Z. Induktive 23. Vesselinovitch. S. D. The Sex Dependent Difference in the Abstammungs-Vererbungslehre,47: 150â€”158,1928. Development of Liver Tumors in Mice Administered 19. Nathanson, L., Hail, T. C., and Farber, S. Biological Aspects of Dimethylnitrosamine. Cancer Res., 29: 1024â€”1027, 1969. Human Malignant Melanoma. Cancer, 20: 650â€”655, 1967.

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Michael J. Siciliano, Alfred Perlmutter and Edward Clark

Cancer Res 1971;31:725-729.

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