Klaus D. Kallman

A New Look at Sex Determination in Poeciliid Fishes

The recent increased interest in sex-determining mechanisms can be traced directly to Wachtel’s concept that the H-Y antigen is the primary testis organizer. There have been several comprehensive reviews dealing with genetic mechanisms of sex determination and sex differentiation, but all of these deal largely with the mammalian literature and pay only lip service to the other classes of vertebrates (J.W. Gordon and Ruddle, 1981; Haseltine and Ohno, 1981; McCarrey and Abbot, 1979; Ohno, 1979; Wachtel and Ohno, 1979). Of particular interest to geneticists are several cases of atypical sex determination in mammals, including humans, in which an autosomal gene causes the testicular differentiation of XX individuals. This has raised the question as to the location of the male-determining gene.

Genetic Control of Gonadotrop Differentiation in the Platyfish, Xiphophorus maculatus (Poeciliidae)

A sex-linked gene controlling the age at which the gonadotrops of the pituitary gland differentiate has been discovered in Xiphophorus maculatus. Males homozygous for early differentiation become sexually mature between 10 and 16 weeks; those homozygous for late maturation between 22 and 40 weeks. Heterozygotes are intermediate. Since growth rate decreases when testes mature. the two classes of males differ significantly in adult size.

The Genus Xiphophorus in Mexico and Central America

The genus Xiphophorus is found from northeastern Mexico (Coahuila) for about 2200 Km as far as Honduras. There are 26 species, of which 21 occupy headwaters on the eastern slope of the Sierra Madre Oriental and continuing Cordillera to the southeast. Virtually all the species in the headwaters occupy limited ranges, often in rivers traversing karst country that are separated from lowland streams by underground passages. Only the three forms in the coastal plain are more widely distributed. Nineteen taxa occur within 400 Km of the Mexican Trans Volcanic Belt, suggesting that the genus may have evolved in this region. In many localities two species are sympatric, but natural hybrids are only known from three or four sites. Four monophyletic groups have been identified: the northern platyfish and the northern swordtail groups, north of the Mexican Trans Volcanic Axis, and to the south the helleri and the clemenciae swordtail groups. The status of the three southern platyfish is still not resolved and the phylogenetic relationship of the different groups to each other is still not fully understood.

Genetics of Melanomas in Xiphophorus Fishes

Abstract Data are presented from studies on heritable melanoma formation in certain hybrids of Xiphophorus fishes. In the best investigated case, benign and malignant melanomas were caused primarily by hemizygous and homozygous loss, respectively, of a tumor suppressor gene that promoted final pigment cell differentiation, although additional genetic events may be of importance. In other melanoma cases, different genes, most of them presumably also tumor suppressor genes, have been implicated in the development of melanomas and may interact at other levels of pigment cell development, such as commitment, migration, or homing of pigment cell precursors. Importantly, genetic factors have been identified that do not allow melanoma formation despite the loss of tumor suppressor genes. Taken together, these findings stress the complexity of tumorigenesis and the heterogeneity of pathways that lead to cancers of the same histological type.

GENETICS OF FIN TRANSPLANTATION IN XIPHOPHORIN FISHES

Successful transplantations of fins between members of one highly inbred strain of platyfish, Xiphophrus maculatus, were shown to be feasible by Kallman and Gordon (1957). Since then, a second highly inbred strain of platyfish was developed in our laboratory that has provided the necessary material for an analysis of the genetic constitution of host and donor in determining whether a graft will be accepted or rejected. The present concept that the fate of a transplant depends upon histocompatibility genes is derived largely from experiments with inbred strains of mice and rats. It is desirable to determine if this genetic concept is applicable to other classes of vertebrates, especially the fishes. This paper is a report of some results that we have obtained thus far from our experiments with the transplantation of fins among members of two highly inbred strains of platyfish and their F1 hybrids.

Continued reproductive potential in aging platyfish as demonstrated by the persistence of gonadotropin, luteinizing hormone releasing hormone and spermatogenesis

Male platyfish, Xiphophorus maculatus, ranging in age from 12 to 54 months (normal life span is 30 months), were examined for age-related changes in the distribution of immunoreactive gonadotropin (ir-GTH) and luteinizing hormone releasing hormone (ir-LHRH) in their pituitary glands, and for changes in the histology of their testes. Males sacrificed at 13 and 16 months of age served as controls. Immunocytochemical methods demonstrate that even in the oldest fish, some almost twice the average platyfish life span, ir-GTH and ir-LHRH are localized in the same pituitary cell types in the caudal pars distalis and pars intermedia as in younger sexually mature fish. The testes of old fish continue to contain all stages of spermatogenesis; however, there are age-related increases in the amount of intertubular connective tissue and the relative number of spermatozeugmata, and distortions in the organization of acini. Our observations indicate that GTH and LHRH production and spermatogenesis continue, and thus a potential for reproductive capability persists, in male platyfish up to the time of their death resulting from old age.

Two New Species of Xiphophorus (Poeciliidae) from the Isthmus of Tehuantepec, Oaxaca, Mexico, with a Discussion of the Distribution of theX. clemenciae Clade

Abstract The swordtail, Xiphophorus clemenciae (Poeciliidae), has been considered a species of special concern because of its apparent limited range. Although described in 1959, it is officially still known only from three locations in the Rio Coatzacoalcos basin, Mexico. Zoogeographic studies have now shown that this species is widespread and abundant but restricted to the uplands of the Rio Coatzacolacos basin where it replaces in many areas the common swordtail, X. helleri. Two new swordtail taxa, X. mixei and X. monticolus, are described from headwater streams of the Rio Jaltepec, a major Rio Coatzacoalcos tributary, Oaxaca, Mexico. The new forms are sympatric in part of their range and replace both X. clemenciae and X. helleri. Morphometric and molecular analyses revealed that X. clemenciae and the two new species constitute a monophyletic clade that exhibits a closer evolutionary affinity to the “northern” swordtails and the “platyfish” group of the genus rather than to X. helleri and the other “southern” swordtails. The evolutionary relationships of these taxa are discussed.

ASSIGNMENT OF AN ERBB-LIKE DNA SEQUENCE TO LINKAGE GROUP VI IN FISHES OF THE GENUS XIPHOPHORUS (POECILIIDAE)

The inheritance of 23 protein polymorphisms was compared with the inheritance of a DNA restriction fragment length polymorphism (RFLP) of a strongly cross-hybridizing erbB-related sequence, epidermal growth factor receptor-like-1 (EGFRL1), in Xiphophorus clemenciae X X. milleri-derived backcross hybrids. Two polymorphic bands were noted in this cross with a v-erbB probe after PstI digestion: a 10-kilobase (kb) band in X. clemenciae and a 9-kb band in X. milleri. Joint segregation analysis of the RFLPs and protein polymorphisms indicate that this erbB-related sequence can be assigned to Xiphophorus linkage group VI, which also comprises genes coding for glutamine synthetase (GLNS), nucleoside phosphorylase-2 (NP2), and transferin (TF). We have designated this RFLP as alleles at a locus called EGFRL1 because of very strong cross-hybridization with the v-erbB probe, known to be homologous to the mammalian EGFR gene. This mapping assignment is the first autosomal linkage of an oncogene sequence reported in fish, which provide a large number of genetically controlled experimental tumor models.

Linkage assignment of a DNA sequence (ERCC2L1) homologous to a human DNA repair gene in Xiphophorus fishes: Implications for the evolutionary derivation of human chromosome 19

Fish gene mapping studies have identified several syntenic groups showing conservation over more than 400 million years of vertebrate evolution. In particular, Xiphophorus linkage group IV has been identified as a homolog of human chromosomes 15 and 19. During mammalian evolution, loci coding for glucosephosphate isomerase, peptidase D, muscle creatine kinase, and several DNA repair genes (ERCC1, ERCC2, and XRCC1) appear as a conserved syntenic group on human chromosome 19. When X. clemenciae and X. milleri PstI endonuclease-digested genomic DNA was used in Southern analysis with a human ERCC2 DNA repair gene probe, a strongly cross-hybridizing restriction fragment length polymorphism was observed. Backcrosses to X. clemenciae from X. milleri x X. clemenciae F1 hybrids allowed tests for linkage of the ERCC2-like polymorphism to markers covering a large proportion of the genome. Statistically significant evidence for linkage was found only for ERCC2L1 and CKM (muscle creatine kinase), with a total of 41 parents and 2 recombinants (4.7% recombination, chi 2 = 35.37, P less than 0.001); no evidence for linkage to GPI and PEPD in linkage group IV was detected. The human chromosome 19 synteny of ERCC2 and CKM thus appears to be conserved in Xiphophorus, while other genes located nearby on human chromosome 19 are in a separate linkage group in this fish. If Xiphophorus gene arrangements prove to be primitive, human chromosome 19 may have arisen from chromosome fusion or translocation events at some point since divergence of mammals and fishes from a common ancestor.

Allele-Specific Marker Generation and Linkage Mapping on the Xiphophorus Sex Chromosomes

There is great interest in the sex chromosomes of Xiphophorus fishes because both WY/YY and XX/XY sex-determining mechanisms function in these species, with at least one taxon possessing all three types of sex chromosomes, and because in certain interspecific hybrids melanoma arises as a consequence of inheritance of the sex-linked macromelanophore determining locus (MDL). Representational difference analysis (RDA) has been used to clone two sequences from the sex-determining region of X. maculatus, including a cholinergic receptor, nicotinic, delta polypeptide (CHRND) orthologue. Allele-specific assays for these sequences, as well as for the sex-linked XMRK1 and XMRK2 genes, were developed to distinguish W, X, and Y chromosomes derived from a X. maculatus (XX/XY) strain and a X. helleri (WY/YY) strain. Linkage mapping localized these markers to linkage group (LG) 24. No recombinants were observed between XMRK2 and MDL, confirming a role for XMRK2 in macromelanophore development. Although the master sex-determining (SD) locus certainly resides on Xiphophorus LG 24, autosomal loci are probably involved in sex determination as well, as indicated by the abnormal sex ratios in the backcross hybrids that contrast theoretical predictions based on LG 24 genotyping. Marker development and allelic discrimination on the Xiphophorus sex chromosomes should prove highly useful for studies that utilize this genus as an animal model.