Fish Physiology and Biochemistry vol. 7 nos 1-4 pp 237-242 (1989) Kugler Publications, Amsterdam/Berkeley
Genetic influences on reproductive system development and function: A review Martin P. Schreibman, Seymour Holtzman and Ronald A. Eckhardt Department of Biology, Brooklyn College of the City University of New York, Brooklyn, New York, U.S.A. 11210 Keywords: genetics, reproduction, fish, aquaculture, aging, maturation, development, endocrinology, brain-pituitary-gonad axis
Abstract This paper presents a current view of the genomic and neuroendocrine interaction based on our studies of the reproductive system in the platyfish (Xiphophorus maculatus). It also presents observations from basic research and applied biologists on natural and artificially reared fishes and indicates that there is a direct genetic involvement in the control of spawning, growth rates, size and age at maturation and final body size, similar to that described in platyfish. The past, present and future association of aquaculture and basic science, especially DNA technology, is discussed and potential directions for future research are presented.
Introduction and discussion The current status of our knowledge of how genetics can be utilized to control reproductive processes in fishes is replete with promise but steeped in limited scientific understanding. It is fitting in the context of this meeting and in the obvious surge of activities related to DNA biotechnology, that we stop to evaluate our position and our directions for further efforts. In this paper we will present an overview and update of what we know about genome and neuroendocrine interaction, essentially derived from our studies in X. maculatus, call attention to the present breach between the aquaculturist and the basic research scientist and provide broad generalizations and pose questions for future research. Fish have been used to study basic genetics for almost 75 years (Gerschler 1914). The platyfish, a viviparous freshwater teleost native to Central America and southern Mexico, has been studied intensively both in the field and in the laboratory by
such eminent investigators as Gordon, Koswig, Anders and Kallman, who have provided much of the seminal and continuing work on this species. Our own studies on the histophysiology of pituitary-regulated physiological processes began in 1960 in collaboration with Klaus Kallman (Schreibman 1964). Since 1971, we have used the platyfish as a model system to study the interaction of genetic and neuroendocrine factors that control the development, maturation and senescence of the brainpituitary-gonadal (BPG) axis. The subject, which has been reviewed recently (Schreibman et al. 1986, 1987), is discussed here in the framework of this conference. A sex-linked gene, P, determines the age at which sexual maturation occurs in male and female platyfish (Kallman and Schreibman 1973; Kallman et al. 1973; Kallman 1975; Schreibman and Kallman 1977). At least 5 P alleles (P' through P5 ) have been identified in natural populations and in laboratory stocks (Kallman and Borkosky 1978). Various combinations of P alleles determine the
238 specific, predictable age at which sexual maturity occurs (anywhere between 8 and 104 weeks of age). Generally speaking, Pl is the allele for the earliest maturation and P5 for later maturation. The various P factors are carried on the sex chromosomes (X and Y) and are closely linked to genes for body pigmentation that serve as their phenotypic markers (Kallman and Borkoski 1978). At the onset of maturation, there is a sequence of developmental events in the BPG axis that is characterized by the sequential appearance of immunoreactive (ir-) gonadotropin releasing hormone (GnRH) in specific centers of the brain, the proliferation of gonadotrops in the caudal pars distalis (CPD) of the pituitary gland and the maturation of the gonads to produce viable gametes. These internal structural and physiological events, which signal the maturation of the reproductive system, are clearly indicated externally in males by the androgen-regulated transformation of the anal fin into a gonopodium (an intromittent organ). The growth rate in males decreases at the time of sexual maturity. Thus, late maturing males are generally larger than early maturing males of the same chronological age. Females of different P genotype continue to grow at a similar rate past puberty and cannot be distinguished by body size (Kallman and Schreibman 1973). Similar sex-linked multiple allelic series at the P locus occur in other species of Xiphophorus (e.g., X. milleri, X. montezumae, X. nigrensis; Kallman 1984). By performing defined genetic crosses, we are capable of producing variations in the offspring of X. maculatus which can be used to address specific questions related to a variety of physiological/ neuroendocrinological processes that are under genetic control. For example, the cross between a X-N X-Sp (P5 PI) female and a X-N Y-Sr (p S p2) male (N, Sp and Sr are genes for nigra, spot-sided and strip-sided pigmentation respectively and are linked to the specific P alleles indicated) yields both early and late maturing brothers and sisters in a single mating. The progeny are easily distinguished by their sex and body pigment patterns. In another cross, males of the Jamapa strain (X-Sp Y-Sr; P'p 2) mated to Belize-Jamapa hybrid females (XSp X-N; P'P 5 ), yield male progeny in the F 1 gener-
ation that mature at two different ages. These males possess the histocompatibility necessary for performing successful tissue transplants. In a third example, the F l progeny resulting from matings within 163A and 163B Jamapa platyfish permit us to compare the age at maturation and maximum life span (see below for discussion). In nine years we have gathered sufficient information to establish the most complete "longitudinal" study available for any cold-blooded vertebrate, and probably for any vertebrate (Schreibman et al. 1985). We have found that the aging of the reproductive system (i.e., from puberty to death) is also clearly under genetic regulation (Schreibman et al. 1987). In adult platyfish, raised under controlled conditions, there are distinct changes in the distribution and relative quantities of neuropeptides, neurotransmitters, pituitary and gonadal hormones as well as in the structure of the cells that manufacture, transport or are the target organs for these factors. These observed changes, which differ in early and late maturing genotypes, are also sexually dimorphic (Schreibman et al. 1985, 1987; Halpern-Sebold et al. 1986; MargolisNunno et al. 1987). As in maturation, it is probable that each GnRH containing region of the brain plays a significant, and perhaps unique, role during the later phases of the life cycle of platyfish (Schreibman et al. 1987). We have also found that in the oldest animals examined, gonads produce gametes and the pituitary gland contains ir-gonadotropin (GTH) and ir-GnRH (Schreibman et al. 1983). Based on recent unpublished observations, it is clear that there is a relationship between P allele constitution and life span. Our observations suggest that the P allele is, or is linked to, the genetic determinant(s) of longevity. We have observed that male and female platyfish which carry the P1 allele, marked by the gene Sp, tend to live longer than genotypes which do not contain PI. Twenty-five percent of those fish that contained the gene P' lived up to, or beyond, 40 months of age. Siblings without the p 1 allele did not survive beyond 34 months of age. The data also suggest that the presence of a functional reproductive system at an early age may be related to longevity because P is
239 also the allele responsible for early sexual maturation (Schreibman and Margolis-Nunno 1988). In the two genetic crosses that follow, we can compare the effects of the chromosome bearing the PI-Sp complex with the effects of the chromosome carrying the P 1-Sd (spotted dorsal fin) complex on longevity. A) 163A PI Fl
Male X-Sd Y-Sr (P'P 2 ) x X-Sd Y-Sr (p l p 2 ) (mature at 10 wks)
Female X-Sd X-Sd (P'P') X-Sd X-Sd (P'Pl) (mature at 8 wks)
Male X-Sp Y-Sr (P'P 2 ) x X-Sp Y-Sr (plp2 ) (mature at 10 wks)
Female X-Sp X-Sp (P I P') X-Sp X-Sp (P'P1) (mature at 8 wks)
B) 163B Pl F1
By comparing the two types of P1 P' females and the two types of p 1p 2 males generated by these two crosses, we could determine whether Pl or the pigment gene associated with it, is more closely associated with longevity. It is essential to determine how the language of the P gene for the calendar of sexual maturation, and perhaps longevity, is translated into the neuroendocrine action that leads to the development, maturation and senescence of the reproductive system. Is the "puberty factor" one and the same with the "aging factor"? The P gene may exert its influence at any level of the BPG axis. One possibility is that the gene may act as a "switch", turning on essential systems for puberty (see Schreibman and Margolis-Nunno 1988). It has also been suggested that the P allele regulates the fate, and not the production, of GnRH (Bao and Kallman 1982). The newest technologies being used in the laboratory may provide more significant ways of extracting the answer to the questions posed above. Most notably, in situ hybridization of nucleic acids permits one to detect and measure specific messenger RNAs (e.g., those directing GnRH synthesis) in individual cells. This technical approach, when couplied with immunocytochemistry, may provide a better idea as to the site(s), time and perhaps the mode of P gene action and activity. Once we know
how the P gene functions in maturation, we may better understand the role of the P gene in the process of senescence. In addition to the material discussed in the previous pages, platyfish have been used specifically to study the mechanisms of sex determination (Kallman 1984), the structural and functional relationships between the olfactory and reproductive systems (Schreibman et al. 1986, 1987; Schreibman and Margolis-Nunno 1987) and the genetic influences on the number and quality of gametes (NN; P5P5 females have fewer and less developed eggs and smaller pituitary glands than siblings of equal size and age but who carry alleles for earlier maturation (Kallman and Schreibman, unpublished observations)). This correlation of genome and physiological activity is certainly not restricted to Xiphophorus. There are numerous observations from basic and applied biologists of both natural and artificially reared species which suggest direct genetic involvement in the control of the time of spawning, growth rates and size and age at maturation, perhaps in a fashion very similar to that described in platyfish. We present here some examples from published accounts to stimulate (and challenge) a re-examination by workers in the field of their specific observations with an eye toward evaluating the genetic involvement of their particular systems:
Salmonids The salmonids have been studied most extensively because of their commercial importance. Salmo gairdneri vary considerably in the proportion of mature to immature individuals in different sibgroups (Moller et al. 1979). It is also clear that the age and size at maturation in commercially important species of salmonids is genetically determined and sexually dimorphic (Donaldson and Benfey 1987; Myers et al. 1986). Early maturing salmonids grow at a faster rate until they reach maturity (Donaldson and Olson 1955; Myers et al. 1986). Selective breeding can result in improved growth rate, earlier maturity, increased tolerance to warmer temperatures, increased egg production, change in spawning season
240 and resistance to disease (Donaldson and Olson 1955). Another parallel to the platyfish is provided by the report that stocks of trout can be distinguished from each other by genetic markers for body coloration, scale patterns and fin morphology (Moav et al. 1978). Furthermore, rainbow trout embryos with a mutant allele (Pgml) that expresses liver phosphoglucomutase activity hatch earlier, begin feeding earlier and retain a size advantage until sexual maturity which tends to occur at an earlier age than sibs without Pgml (Allendorf et al. 1983).
Cyprinids The European and Chinese races of the common carp differ significantly in growth rate, viability, fertility, age at sexual maturation, weight and size (Wohlfarth et al. 1975). However, sex differences in growth rate are not consistent from species to species (e.g., male tilapia and female salmonids grow faster; Reay 1984).
Other species In gobioid fishes the age at sexual maturity ranges from a few months to at least three years (Miller 1984; Mann et al. 1984; Staples 1975); in herrings, two main groups have been identified in the North Sea that spawn at different times and at different locations (Iles 1984); and in redfishes (Sebastes), the difference in the size and age at maturation in the Northwest Atlantic may have a genetic and sexually dimorphic basis (Ni and Sandeman 1984). It is unfortunate that the just described genetic correlates of the physiological factors that influence reproduction are not fully appreciated; they should receive greater attention. A better understanding is certain to lead to methods that will improve aquaculture. The ancient art of aquaculture and the not so ancient art of DNA technology have followed divergent paths. Carp have been cultivated for three to four thousand years in China and for 600 years in Europe (even goldfish, the aquatic laboratory rat, has been domesticated for more than one thousand years), and yet compared to the
revolutionary changes in agriculture, progress and technological improvement have been slow in coming. On the other hand, the study of gene expression per se began some twenty years ago. The origin of DNA technology probably lies in the classical paper in which Avery et al. (1944) reported genetic transformation in bacteria resulting from adding DNA to their culture media. The development of procedures to improve aquaculture has, until recently, been approached essentially by trial and error; more scientific approaches should permit more rapid advancement. The most frequently used method for controlling the quality and quantity of fish under artificial growth conditions include: (1) the selection and calculated breeding, including selection and storing of desirable gametes containing favored genetic factors; (2) the introduction of favored segments of chromosomes after separation and cloning; (3) the masking of the effect of genome by artificial control of photoperiod, temperature, nutrition and hormone levels; (4) the selection for fitness (which includes fecundity, longevity, and number of spawnings); and (5) the selection of gender that are faster growing or differ in their age at maturation. The ability to regulate the growth and reproductive patterns of broodstock and their offspring offers limitless potential and benefits for the aquaculturist. Only by understanding the biological and physiological bases of these phenomena will we be successful in learning how to strictly control the farming of desirable species and, perhaps, deal more effectively with the problems of conservation. As an example, excessive fishing over the years has had tremendous effects on natural selection. The intensification of fishing for Tilapia nilotica in Lake George, Africa since 1950 has resulted in a decrease of the mean size of the fish caught from 1900 to 400 g and the length at maturity has decreased from 29 to 18 cm (J.J. Gwahaba 1937, cited in Moav et al. 1978). It also appears to provide another example that there may be at least two populations in wild species of early and late maturing variants. It is important to evaluate breeding experiments with care. Verspoor (1988) has described the deleterious effects of excessive hybridization in nature
241 when a new species enters the range of another by either natural events or artificial introduction. By contrast, Burns and Kallman (1985) have reported the deleterious effects of inbred genetic stocks of Xiphophorus, but they have also noted that some strains, even after more than 60 generations of sibling matings, are equal to wild fish in reproductive performance. We need to increase our understanding of environmental, nutritional and genetic influences on reproduction, and to transfer manipulative techniques developed in the laboratory to practical use in the ponds. The convergence of aquaculture and DNA technologies is certain to have tremendous impact on the fundamental and applied aspects of the aquatic sciences. It is clear from the reports at this meeting that we are on the threshold of a very exciting period in which newly developed methods for the manipulation, handling and transferring of genes will have tremendous repercussions on the improvement of aquaculture technology.
Acknowledgements The research, which provided much of the basic information on platyfish reported in this paper, has been supported over the years by grants from the NSF (PCM77-15981), the NIH-NIA (AGO-1938), The City University of New York (PSC-CUNY), NATO (333/84) and BARD (1-772-84). We thank Ms. Ethel Suben for many years of devoted and professional technical assistance.
References cited Allendorf, F.W., Knudsen, K.L. and Leary, R.F. 1983. Adaptive significance of differences in the tissue specific expression of a phosphoglucomutase gene in rainbow trout. Proc. Nat. Acad. Sci. U.S.A. 80: 1397-1400. Avery, O.T., MacLeod, C.M. and McCarty, M. 1944. Studies on the nature of the substance inducing transformation by an desoxyribonucleic acid fraction isolated from Pneumococcus type III. J. Exp. Med. 79: 137-157. Burns, J.R. and Kallman, K.D. 1985. An ovarian regression syndrome in the platyfish, Xiphophorus maculatus. J. Exp. Zool. 233: 301-316. Bao, I.Y. and Kallman, K.D. 1982. Genetic control of the
hypothalamopituitary axis and the effect of hybridization on sexual maturation (Xiphophorus, Pisces Poeciliidae). J. Exp. Zool. 200: 297-309. Donaldson, E.M. and Benfey, T.J. 1987. Current status of sex manipulation. In Proc. Third Intl. Symp. Reprod. Physiol. of Fish. pp. 108-119. Edited by D.R. Idler, L. Crim and J. Walsh. Memorial University Press, St. John's. Donaldson, L.R. and Olson, P.R. 1955. Development of rainbow trout brood stock by selective breeding. Trans. Am. Fish. Soc. 85: 93-101. Gerschler, M.W. 1914. Uber Alternative Vererbung bei Kreuving von Cyprinodontiden-Gattungen. Z. Ind. Abst. Vererbgsl. 12: 73-96. Halpern-Sebold, L., Schreibman, M.P. and Margolis-Nunno, H. 1986. Differences between early- and late-maturing genotypes of the platyfish (Xiphophorus maculatus) in the morphometry of their immunoreactive LHRH containing cells: A developmental study. J. Exp. Zool. 240: 245-257. Iles, T.D. 1984. Allocation of resources to gonad and soma in Atlantic herring. In Fish Reproduction: Strategies and Tactics. pp. 331-347. Edited by Potts, G.W. and Wootton, R.J. Academic Press, New York. Kallman, K.D. 1975. The platyfish, Xiphophorusmaculatus. In Handbook of Genetics. pp. 81-132. Edited by R.C. King. Plenum Publ. Corp., New York. Kallman, K.D. 1984. A new look at sex determination in Poeciliid fishes. In Evolutionary Genetics of Fishes. pp. 95-171. Edited by B.J. Turner. Plenum Publ. Corp., New York. Kallman, K.D. and Borkoski, V. 1978. A sex-linked gene controlling the onset of sexual maturation in female and male platyfish (Xiphophorus maculatus), fecundity in females and adult size in males. Genetics 89: 79-119. Kallman, K.D. and Schreibman, M.P. 1973. A sex-linked gene controlling gonadotrop differentiation and its significance in determining the age of sexual maturation and size of the platyfish, Xiphophorus maculatus. Gen. Comp. Endocrinol. 21: 287-304. Kallman, K.D., Schreibman, M.P. and Borkoski, V. 1973. Genetic control of gonadotrop differentiation in the platyfish, Xiphophorus maculatus (Poeciliidae). Science 58: 678-680. Mann, R.H.K., Mills, C.A. and Crisp, D.T. 1984. Geographical variation in the life-history tactics of some species of freshwater fish. In Fish Reproduction: Strategies and Tactics. pp. 171-186. Edited by G.W. Potts and R.J. Wootton. Academic Press, New York. Margolis-Nunno, H., Schreibman, M.P. and Halpern-Sebold, L. 1987. Sexually dimorphic age-related differences in the distribution of somatostatin in the platyfish. Mech. Aging Develop. 41: 139-148. Miller, P.J. 1984. The tokology of Gobioid fishes. In Fish Reproduction: Strategies and Tactics. pp. 119-154. Edited by G.W. Potts and R.J Wootton. Academic Press, New York. Moav, R., Brody, T. and Hulata, G. 1978. Genetic improvement of wild fish populations. Science 201: 1090-1094.
242 Moller, D., Naevdal, G., Holm, M. and Leroy, R. 1979. In Advances in Aquaculture. pp. 622-625. Edited by T.V.R. Pillay and W.E. Dill. Fishery News Books, London. Myers, R.A., Hutchings, J.A. and Gibson, R.J. 1986. Variation in male parr maturation within and among populations of Atlantic salmon, Salmo salar. Can. J. Fish. Aquat. Sci. 43: 1242-1248. Ni, I.-H. and Sandeman, E.J. 1984. Size at maturity for northwest Atlantic redfishes (Sebastes). Can. J. Fish. Aquat. Sci. 41: 1753-1762. Pickett, M.H., Hew, C.L. and Davies, P.L. 1983. Seasonal variation in the level of antifreeze protein mRNA from the winter flounder. Biochim. Biophys. Acta 739: 97-104. Reay, P.J. 1984. Reproductive tactics: A non-event in aquaculture? In Fish Reproduction: Strategies and Tactics. pp. 291-309. Edited by G.W. Potts and R.J. Wootton. Academic Press, New York. Schreibman, M.P. 1964. Studies on the pituitary gland of Xiphophorus maculatus (the platyfish). Zoologica 49: 217-243. Schreibman, M.P., Halpern-Sebold, L. and Margolis-Nunno, H. 1985. Sexually dimorphic, age-related changes in the distribution of immunoreactive LHRH in platyfish. Mech. Aging Develop. 33: 29-37. Schreibman, M.P. and Kallman, K.D. 1977. The genetic control of the pituitary-gonadal axis in the platyfish, Xiphophorus maculatus. J. Exp. Zool. 200: 277-294. Schreibman, M.P., Margolis-Kazan, H., Bloom, J.L. and Kallman, K.D. 1983. Continued reproductive potential in aging platyfish as demonstrated by the persistence of gonadotropin, luteinizing hormone releasing hormone and
spermatogenesis. Mech. Aging Develop. 22: 105-112. Schreibman, M.P. and Margolis-Nunno, H. 1987. Reproductive biology of the terminal nerve (nucleus olfactoretinalis) and other LHRH pathways in teleost fishes. Ann. N.Y. Acad. Sci. 519: 60-68. Schreibman, M.P. and Margolis-Nunno, H. 1989. Aging of the brain-pituitary-gonadal axis in poikilotherms. In Aging, Development and Senescence of Vertebrate Neuroendocrine Systems. Edited by M.P. Schreibman and C.P. Scanes. Academic Press, New York. (In press). Schreibman, M.P., Margolis-Nunno, H. and Halpern-Sebold, L. 1986. The structural and functional relationships between olfactory and reproductive systems from birth to old age in fish. In Signals in Vertebrates. Vol. IV. pp. 155-172. Edited by N.D. Duval, D. Muller-Schwarze and R.M Silverstein. Plenum Press, New York. Schreibman, M.P., Margolis-Nunno, H. and Halpern-Sebold, L. 1987. Aging of the neuroendocrine system. In Hormones and Reproduction in Fishes, Amphibians, and Reptiles. pp. 563-584. Edited by D.O. Norris and R.E. Jones. Plenum Press Publ. Corp., New York. Staples, D.J. 1975. Production biology of the upland bully Philypnodon breviceps in a small New Zealand lake. I. Life history, food, feeding and activity rhythms. J. Fish Biol. 7: 1-24. Verspoor, E. 1988. Widespread hybridization between Atlantic salmon, Salmo salar, and introduced brown trout, S. trutta, in eastern Newfoundland. J. Fish Biol. 32: 327-334. Wohlfarth, G., Moav, R. and Hulata, G. 1975. Genetic differences between the Chinese and European races of the common carp. Heredity 34: 341-350.
Genetic influences on reproductive system development and function: A review Martin P. Schreibman, Seymour Holtzman and Ronald A. Eckhardt Department of Biology, Brooklyn College of the City University of New York, Brooklyn, New York, U.S.A. 11210 Keywords: genetics, reproduction, fish, aquaculture, aging, maturation, development, endocrinology, brain-pituitary-gonad axis
Abstract This paper presents a current view of the genomic and neuroendocrine interaction based on our studies of the reproductive system in the platyfish (Xiphophorus maculatus). It also presents observations from basic research and applied biologists on natural and artificially reared fishes and indicates that there is a direct genetic involvement in the control of spawning, growth rates, size and age at maturation and final body size, similar to that described in platyfish. The past, present and future association of aquaculture and basic science, especially DNA technology, is discussed and potential directions for future research are presented.
Introduction and discussion The current status of our knowledge of how genetics can be utilized to control reproductive processes in fishes is replete with promise but steeped in limited scientific understanding. It is fitting in the context of this meeting and in the obvious surge of activities related to DNA biotechnology, that we stop to evaluate our position and our directions for further efforts. In this paper we will present an overview and update of what we know about genome and neuroendocrine interaction, essentially derived from our studies in X. maculatus, call attention to the present breach between the aquaculturist and the basic research scientist and provide broad generalizations and pose questions for future research. Fish have been used to study basic genetics for almost 75 years (Gerschler 1914). The platyfish, a viviparous freshwater teleost native to Central America and southern Mexico, has been studied intensively both in the field and in the laboratory by
such eminent investigators as Gordon, Koswig, Anders and Kallman, who have provided much of the seminal and continuing work on this species. Our own studies on the histophysiology of pituitary-regulated physiological processes began in 1960 in collaboration with Klaus Kallman (Schreibman 1964). Since 1971, we have used the platyfish as a model system to study the interaction of genetic and neuroendocrine factors that control the development, maturation and senescence of the brainpituitary-gonadal (BPG) axis. The subject, which has been reviewed recently (Schreibman et al. 1986, 1987), is discussed here in the framework of this conference. A sex-linked gene, P, determines the age at which sexual maturation occurs in male and female platyfish (Kallman and Schreibman 1973; Kallman et al. 1973; Kallman 1975; Schreibman and Kallman 1977). At least 5 P alleles (P' through P5 ) have been identified in natural populations and in laboratory stocks (Kallman and Borkosky 1978). Various combinations of P alleles determine the
238 specific, predictable age at which sexual maturity occurs (anywhere between 8 and 104 weeks of age). Generally speaking, Pl is the allele for the earliest maturation and P5 for later maturation. The various P factors are carried on the sex chromosomes (X and Y) and are closely linked to genes for body pigmentation that serve as their phenotypic markers (Kallman and Borkoski 1978). At the onset of maturation, there is a sequence of developmental events in the BPG axis that is characterized by the sequential appearance of immunoreactive (ir-) gonadotropin releasing hormone (GnRH) in specific centers of the brain, the proliferation of gonadotrops in the caudal pars distalis (CPD) of the pituitary gland and the maturation of the gonads to produce viable gametes. These internal structural and physiological events, which signal the maturation of the reproductive system, are clearly indicated externally in males by the androgen-regulated transformation of the anal fin into a gonopodium (an intromittent organ). The growth rate in males decreases at the time of sexual maturity. Thus, late maturing males are generally larger than early maturing males of the same chronological age. Females of different P genotype continue to grow at a similar rate past puberty and cannot be distinguished by body size (Kallman and Schreibman 1973). Similar sex-linked multiple allelic series at the P locus occur in other species of Xiphophorus (e.g., X. milleri, X. montezumae, X. nigrensis; Kallman 1984). By performing defined genetic crosses, we are capable of producing variations in the offspring of X. maculatus which can be used to address specific questions related to a variety of physiological/ neuroendocrinological processes that are under genetic control. For example, the cross between a X-N X-Sp (P5 PI) female and a X-N Y-Sr (p S p2) male (N, Sp and Sr are genes for nigra, spot-sided and strip-sided pigmentation respectively and are linked to the specific P alleles indicated) yields both early and late maturing brothers and sisters in a single mating. The progeny are easily distinguished by their sex and body pigment patterns. In another cross, males of the Jamapa strain (X-Sp Y-Sr; P'p 2) mated to Belize-Jamapa hybrid females (XSp X-N; P'P 5 ), yield male progeny in the F 1 gener-
ation that mature at two different ages. These males possess the histocompatibility necessary for performing successful tissue transplants. In a third example, the F l progeny resulting from matings within 163A and 163B Jamapa platyfish permit us to compare the age at maturation and maximum life span (see below for discussion). In nine years we have gathered sufficient information to establish the most complete "longitudinal" study available for any cold-blooded vertebrate, and probably for any vertebrate (Schreibman et al. 1985). We have found that the aging of the reproductive system (i.e., from puberty to death) is also clearly under genetic regulation (Schreibman et al. 1987). In adult platyfish, raised under controlled conditions, there are distinct changes in the distribution and relative quantities of neuropeptides, neurotransmitters, pituitary and gonadal hormones as well as in the structure of the cells that manufacture, transport or are the target organs for these factors. These observed changes, which differ in early and late maturing genotypes, are also sexually dimorphic (Schreibman et al. 1985, 1987; Halpern-Sebold et al. 1986; MargolisNunno et al. 1987). As in maturation, it is probable that each GnRH containing region of the brain plays a significant, and perhaps unique, role during the later phases of the life cycle of platyfish (Schreibman et al. 1987). We have also found that in the oldest animals examined, gonads produce gametes and the pituitary gland contains ir-gonadotropin (GTH) and ir-GnRH (Schreibman et al. 1983). Based on recent unpublished observations, it is clear that there is a relationship between P allele constitution and life span. Our observations suggest that the P allele is, or is linked to, the genetic determinant(s) of longevity. We have observed that male and female platyfish which carry the P1 allele, marked by the gene Sp, tend to live longer than genotypes which do not contain PI. Twenty-five percent of those fish that contained the gene P' lived up to, or beyond, 40 months of age. Siblings without the p 1 allele did not survive beyond 34 months of age. The data also suggest that the presence of a functional reproductive system at an early age may be related to longevity because P is
239 also the allele responsible for early sexual maturation (Schreibman and Margolis-Nunno 1988). In the two genetic crosses that follow, we can compare the effects of the chromosome bearing the PI-Sp complex with the effects of the chromosome carrying the P 1-Sd (spotted dorsal fin) complex on longevity. A) 163A PI Fl
Male X-Sd Y-Sr (P'P 2 ) x X-Sd Y-Sr (p l p 2 ) (mature at 10 wks)
Female X-Sd X-Sd (P'P') X-Sd X-Sd (P'Pl) (mature at 8 wks)
Male X-Sp Y-Sr (P'P 2 ) x X-Sp Y-Sr (plp2 ) (mature at 10 wks)
Female X-Sp X-Sp (P I P') X-Sp X-Sp (P'P1) (mature at 8 wks)
B) 163B Pl F1
By comparing the two types of P1 P' females and the two types of p 1p 2 males generated by these two crosses, we could determine whether Pl or the pigment gene associated with it, is more closely associated with longevity. It is essential to determine how the language of the P gene for the calendar of sexual maturation, and perhaps longevity, is translated into the neuroendocrine action that leads to the development, maturation and senescence of the reproductive system. Is the "puberty factor" one and the same with the "aging factor"? The P gene may exert its influence at any level of the BPG axis. One possibility is that the gene may act as a "switch", turning on essential systems for puberty (see Schreibman and Margolis-Nunno 1988). It has also been suggested that the P allele regulates the fate, and not the production, of GnRH (Bao and Kallman 1982). The newest technologies being used in the laboratory may provide more significant ways of extracting the answer to the questions posed above. Most notably, in situ hybridization of nucleic acids permits one to detect and measure specific messenger RNAs (e.g., those directing GnRH synthesis) in individual cells. This technical approach, when couplied with immunocytochemistry, may provide a better idea as to the site(s), time and perhaps the mode of P gene action and activity. Once we know
how the P gene functions in maturation, we may better understand the role of the P gene in the process of senescence. In addition to the material discussed in the previous pages, platyfish have been used specifically to study the mechanisms of sex determination (Kallman 1984), the structural and functional relationships between the olfactory and reproductive systems (Schreibman et al. 1986, 1987; Schreibman and Margolis-Nunno 1987) and the genetic influences on the number and quality of gametes (NN; P5P5 females have fewer and less developed eggs and smaller pituitary glands than siblings of equal size and age but who carry alleles for earlier maturation (Kallman and Schreibman, unpublished observations)). This correlation of genome and physiological activity is certainly not restricted to Xiphophorus. There are numerous observations from basic and applied biologists of both natural and artificially reared species which suggest direct genetic involvement in the control of the time of spawning, growth rates and size and age at maturation, perhaps in a fashion very similar to that described in platyfish. We present here some examples from published accounts to stimulate (and challenge) a re-examination by workers in the field of their specific observations with an eye toward evaluating the genetic involvement of their particular systems:
Salmonids The salmonids have been studied most extensively because of their commercial importance. Salmo gairdneri vary considerably in the proportion of mature to immature individuals in different sibgroups (Moller et al. 1979). It is also clear that the age and size at maturation in commercially important species of salmonids is genetically determined and sexually dimorphic (Donaldson and Benfey 1987; Myers et al. 1986). Early maturing salmonids grow at a faster rate until they reach maturity (Donaldson and Olson 1955; Myers et al. 1986). Selective breeding can result in improved growth rate, earlier maturity, increased tolerance to warmer temperatures, increased egg production, change in spawning season
240 and resistance to disease (Donaldson and Olson 1955). Another parallel to the platyfish is provided by the report that stocks of trout can be distinguished from each other by genetic markers for body coloration, scale patterns and fin morphology (Moav et al. 1978). Furthermore, rainbow trout embryos with a mutant allele (Pgml) that expresses liver phosphoglucomutase activity hatch earlier, begin feeding earlier and retain a size advantage until sexual maturity which tends to occur at an earlier age than sibs without Pgml (Allendorf et al. 1983).
Cyprinids The European and Chinese races of the common carp differ significantly in growth rate, viability, fertility, age at sexual maturation, weight and size (Wohlfarth et al. 1975). However, sex differences in growth rate are not consistent from species to species (e.g., male tilapia and female salmonids grow faster; Reay 1984).
Other species In gobioid fishes the age at sexual maturity ranges from a few months to at least three years (Miller 1984; Mann et al. 1984; Staples 1975); in herrings, two main groups have been identified in the North Sea that spawn at different times and at different locations (Iles 1984); and in redfishes (Sebastes), the difference in the size and age at maturation in the Northwest Atlantic may have a genetic and sexually dimorphic basis (Ni and Sandeman 1984). It is unfortunate that the just described genetic correlates of the physiological factors that influence reproduction are not fully appreciated; they should receive greater attention. A better understanding is certain to lead to methods that will improve aquaculture. The ancient art of aquaculture and the not so ancient art of DNA technology have followed divergent paths. Carp have been cultivated for three to four thousand years in China and for 600 years in Europe (even goldfish, the aquatic laboratory rat, has been domesticated for more than one thousand years), and yet compared to the
revolutionary changes in agriculture, progress and technological improvement have been slow in coming. On the other hand, the study of gene expression per se began some twenty years ago. The origin of DNA technology probably lies in the classical paper in which Avery et al. (1944) reported genetic transformation in bacteria resulting from adding DNA to their culture media. The development of procedures to improve aquaculture has, until recently, been approached essentially by trial and error; more scientific approaches should permit more rapid advancement. The most frequently used method for controlling the quality and quantity of fish under artificial growth conditions include: (1) the selection and calculated breeding, including selection and storing of desirable gametes containing favored genetic factors; (2) the introduction of favored segments of chromosomes after separation and cloning; (3) the masking of the effect of genome by artificial control of photoperiod, temperature, nutrition and hormone levels; (4) the selection for fitness (which includes fecundity, longevity, and number of spawnings); and (5) the selection of gender that are faster growing or differ in their age at maturation. The ability to regulate the growth and reproductive patterns of broodstock and their offspring offers limitless potential and benefits for the aquaculturist. Only by understanding the biological and physiological bases of these phenomena will we be successful in learning how to strictly control the farming of desirable species and, perhaps, deal more effectively with the problems of conservation. As an example, excessive fishing over the years has had tremendous effects on natural selection. The intensification of fishing for Tilapia nilotica in Lake George, Africa since 1950 has resulted in a decrease of the mean size of the fish caught from 1900 to 400 g and the length at maturity has decreased from 29 to 18 cm (J.J. Gwahaba 1937, cited in Moav et al. 1978). It also appears to provide another example that there may be at least two populations in wild species of early and late maturing variants. It is important to evaluate breeding experiments with care. Verspoor (1988) has described the deleterious effects of excessive hybridization in nature
241 when a new species enters the range of another by either natural events or artificial introduction. By contrast, Burns and Kallman (1985) have reported the deleterious effects of inbred genetic stocks of Xiphophorus, but they have also noted that some strains, even after more than 60 generations of sibling matings, are equal to wild fish in reproductive performance. We need to increase our understanding of environmental, nutritional and genetic influences on reproduction, and to transfer manipulative techniques developed in the laboratory to practical use in the ponds. The convergence of aquaculture and DNA technologies is certain to have tremendous impact on the fundamental and applied aspects of the aquatic sciences. It is clear from the reports at this meeting that we are on the threshold of a very exciting period in which newly developed methods for the manipulation, handling and transferring of genes will have tremendous repercussions on the improvement of aquaculture technology.
Acknowledgements The research, which provided much of the basic information on platyfish reported in this paper, has been supported over the years by grants from the NSF (PCM77-15981), the NIH-NIA (AGO-1938), The City University of New York (PSC-CUNY), NATO (333/84) and BARD (1-772-84). We thank Ms. Ethel Suben for many years of devoted and professional technical assistance.
References cited Allendorf, F.W., Knudsen, K.L. and Leary, R.F. 1983. Adaptive significance of differences in the tissue specific expression of a phosphoglucomutase gene in rainbow trout. Proc. Nat. Acad. Sci. U.S.A. 80: 1397-1400. Avery, O.T., MacLeod, C.M. and McCarty, M. 1944. Studies on the nature of the substance inducing transformation by an desoxyribonucleic acid fraction isolated from Pneumococcus type III. J. Exp. Med. 79: 137-157. Burns, J.R. and Kallman, K.D. 1985. An ovarian regression syndrome in the platyfish, Xiphophorus maculatus. J. Exp. Zool. 233: 301-316. Bao, I.Y. and Kallman, K.D. 1982. Genetic control of the
hypothalamopituitary axis and the effect of hybridization on sexual maturation (Xiphophorus, Pisces Poeciliidae). J. Exp. Zool. 200: 297-309. Donaldson, E.M. and Benfey, T.J. 1987. Current status of sex manipulation. In Proc. Third Intl. Symp. Reprod. Physiol. of Fish. pp. 108-119. Edited by D.R. Idler, L. Crim and J. Walsh. Memorial University Press, St. John's. Donaldson, L.R. and Olson, P.R. 1955. Development of rainbow trout brood stock by selective breeding. Trans. Am. Fish. Soc. 85: 93-101. Gerschler, M.W. 1914. Uber Alternative Vererbung bei Kreuving von Cyprinodontiden-Gattungen. Z. Ind. Abst. Vererbgsl. 12: 73-96. Halpern-Sebold, L., Schreibman, M.P. and Margolis-Nunno, H. 1986. Differences between early- and late-maturing genotypes of the platyfish (Xiphophorus maculatus) in the morphometry of their immunoreactive LHRH containing cells: A developmental study. J. Exp. Zool. 240: 245-257. Iles, T.D. 1984. Allocation of resources to gonad and soma in Atlantic herring. In Fish Reproduction: Strategies and Tactics. pp. 331-347. Edited by Potts, G.W. and Wootton, R.J. Academic Press, New York. Kallman, K.D. 1975. The platyfish, Xiphophorusmaculatus. In Handbook of Genetics. pp. 81-132. Edited by R.C. King. Plenum Publ. Corp., New York. Kallman, K.D. 1984. A new look at sex determination in Poeciliid fishes. In Evolutionary Genetics of Fishes. pp. 95-171. Edited by B.J. Turner. Plenum Publ. Corp., New York. Kallman, K.D. and Borkoski, V. 1978. A sex-linked gene controlling the onset of sexual maturation in female and male platyfish (Xiphophorus maculatus), fecundity in females and adult size in males. Genetics 89: 79-119. Kallman, K.D. and Schreibman, M.P. 1973. A sex-linked gene controlling gonadotrop differentiation and its significance in determining the age of sexual maturation and size of the platyfish, Xiphophorus maculatus. Gen. Comp. Endocrinol. 21: 287-304. Kallman, K.D., Schreibman, M.P. and Borkoski, V. 1973. Genetic control of gonadotrop differentiation in the platyfish, Xiphophorus maculatus (Poeciliidae). Science 58: 678-680. Mann, R.H.K., Mills, C.A. and Crisp, D.T. 1984. Geographical variation in the life-history tactics of some species of freshwater fish. In Fish Reproduction: Strategies and Tactics. pp. 171-186. Edited by G.W. Potts and R.J. Wootton. Academic Press, New York. Margolis-Nunno, H., Schreibman, M.P. and Halpern-Sebold, L. 1987. Sexually dimorphic age-related differences in the distribution of somatostatin in the platyfish. Mech. Aging Develop. 41: 139-148. Miller, P.J. 1984. The tokology of Gobioid fishes. In Fish Reproduction: Strategies and Tactics. pp. 119-154. Edited by G.W. Potts and R.J Wootton. Academic Press, New York. Moav, R., Brody, T. and Hulata, G. 1978. Genetic improvement of wild fish populations. Science 201: 1090-1094.
242 Moller, D., Naevdal, G., Holm, M. and Leroy, R. 1979. In Advances in Aquaculture. pp. 622-625. Edited by T.V.R. Pillay and W.E. Dill. Fishery News Books, London. Myers, R.A., Hutchings, J.A. and Gibson, R.J. 1986. Variation in male parr maturation within and among populations of Atlantic salmon, Salmo salar. Can. J. Fish. Aquat. Sci. 43: 1242-1248. Ni, I.-H. and Sandeman, E.J. 1984. Size at maturity for northwest Atlantic redfishes (Sebastes). Can. J. Fish. Aquat. Sci. 41: 1753-1762. Pickett, M.H., Hew, C.L. and Davies, P.L. 1983. Seasonal variation in the level of antifreeze protein mRNA from the winter flounder. Biochim. Biophys. Acta 739: 97-104. Reay, P.J. 1984. Reproductive tactics: A non-event in aquaculture? In Fish Reproduction: Strategies and Tactics. pp. 291-309. Edited by G.W. Potts and R.J. Wootton. Academic Press, New York. Schreibman, M.P. 1964. Studies on the pituitary gland of Xiphophorus maculatus (the platyfish). Zoologica 49: 217-243. Schreibman, M.P., Halpern-Sebold, L. and Margolis-Nunno, H. 1985. Sexually dimorphic, age-related changes in the distribution of immunoreactive LHRH in platyfish. Mech. Aging Develop. 33: 29-37. Schreibman, M.P. and Kallman, K.D. 1977. The genetic control of the pituitary-gonadal axis in the platyfish, Xiphophorus maculatus. J. Exp. Zool. 200: 277-294. Schreibman, M.P., Margolis-Kazan, H., Bloom, J.L. and Kallman, K.D. 1983. Continued reproductive potential in aging platyfish as demonstrated by the persistence of gonadotropin, luteinizing hormone releasing hormone and
spermatogenesis. Mech. Aging Develop. 22: 105-112. Schreibman, M.P. and Margolis-Nunno, H. 1987. Reproductive biology of the terminal nerve (nucleus olfactoretinalis) and other LHRH pathways in teleost fishes. Ann. N.Y. Acad. Sci. 519: 60-68. Schreibman, M.P. and Margolis-Nunno, H. 1989. Aging of the brain-pituitary-gonadal axis in poikilotherms. In Aging, Development and Senescence of Vertebrate Neuroendocrine Systems. Edited by M.P. Schreibman and C.P. Scanes. Academic Press, New York. (In press). Schreibman, M.P., Margolis-Nunno, H. and Halpern-Sebold, L. 1986. The structural and functional relationships between olfactory and reproductive systems from birth to old age in fish. In Signals in Vertebrates. Vol. IV. pp. 155-172. Edited by N.D. Duval, D. Muller-Schwarze and R.M Silverstein. Plenum Press, New York. Schreibman, M.P., Margolis-Nunno, H. and Halpern-Sebold, L. 1987. Aging of the neuroendocrine system. In Hormones and Reproduction in Fishes, Amphibians, and Reptiles. pp. 563-584. Edited by D.O. Norris and R.E. Jones. Plenum Press Publ. Corp., New York. Staples, D.J. 1975. Production biology of the upland bully Philypnodon breviceps in a small New Zealand lake. I. Life history, food, feeding and activity rhythms. J. Fish Biol. 7: 1-24. Verspoor, E. 1988. Widespread hybridization between Atlantic salmon, Salmo salar, and introduced brown trout, S. trutta, in eastern Newfoundland. J. Fish Biol. 32: 327-334. Wohlfarth, G., Moav, R. and Hulata, G. 1975. Genetic differences between the Chinese and European races of the common carp. Heredity 34: 341-350.
