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.

A Sex-Linked Gene Controlling the Onset of Sexual Maturity in Female and Male Platyfish (XIPHOPHORUS MACULATUS), Fecundity in Females and Adult Size in Males.

A sex-linked gene, P, controls the onset of sexual maturity in the platyfish, Xiphophorus maculatus. The activity of this gene is correlated with the age and size at which the gonadotropic zone of the adenohypophysis differentiates and becomes physiologically active. Immature fish of all genotypes grow at the same rate; however, as adults, males with "early" genotypes are significantly smaller than males of "late" genotypes, since growth rate declines strongly under the influence of androgenic hormone. Five alleles, P(1)... P(5), have been identified from natural populations that under controlled conditions cause gonad maturation between eight and 73 weeks. P(1)P(1) males become mature at eight weeks and 21 mm, P(2)P(2) and P(3)P(3) males between eleven and 13.5 weeks and 25 to 29 mm, and P(4)P(4) males at 25 weeks and 37 mm. Since P(5) is X-linked, no males homozygous for P(5) could be produced. The difference between P(2) and P(3) is largely based upon their interaction with P(5). P(3)P(5) males mature at 17.5 weeks and 33.5 mm and P(2)P(5) males at 28 weeks and 38 mm. The rate of transformation of the unmodified anal fin into a gonopodium, which is under androgenic control, is directly related to the age at initiation of sexual maturity, ranging from 3.2 weeks in P(1)P(1) males to seven weeks in P(2)P( 5) males. These differences may reflect different levels of circulating gonadotropic and androgenic hormones.-In two genotypes of females, initiation of vitellogenesis was closely correlated with size and this critical size was independent of age (e.g., 21 mm for P(1)P(1 )). In a third genotype (P(1)P(5)) the minimum size for vitellogenesis decreased with increasing age, so that females would mature as early as eleven weeks, provided they had attained at least 29 mm, but at 25 weeks even females as small as 23 mm possessed ripe gonads. For P(5)P(5) females, which become mature between 34 and 73 weeks of age, there is no correlation between size and initiation of vitellogenesis. In all four genotypes of females examined, egg number is strongly correlated with size, but the regression of egg number on standard length is distinct for each genotype. Late maturation of P(5)P( 5) females is not offset by an increased number of eggs; for this genotype there is a strong negative correlation between age and number of eggs. Heterozygous fish always mature later than those homozygous for the "earlier" allele. The site of action of the P locus could be the pituitary gland, the hypothalamus or higher centers of the brain where peripheral information is transduced into an appropriate signal required for the activation of the hypothalamus-pituitary-gonadal axis. The P gene could also control the peripheral information. The platyfish may be a useful model to test theories concerning the evolution of life history strategies.

Genetic linkage map of fishes of the genus Xiphophorus (Teleostei: Poeciliidae).

Analysis of genotypes of 76 polymorphic loci in more than 2600 backcross hybrid individuals derived from intra- and interspecific genetic crosses of fishes of the genus Xiphophorus (Poeciliidae) resulted in the identification of 17 multipoint linkage groups containing 55 protein-coding loci and one sex chromosome-linked pigment pattern gene. Multipoint linkage analyses identified highly probable gene orders for 10 linkage groups. The total genome length was estimated to be approximately 18 Morgans. Comparisons of the Xiphophorus linkage map with those of other fishes, amphibians and mammals suggested that fish gene maps are remarkably similar and probably retain many syntenic groups present in the ancestor of all vertebrates.

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.

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.

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.

Female heterogamety in the swordtail, Xiphophorus alvarezi Rosen (Pisces, Poeciliidae), with comments on a natural polymorphism affecting sword coloration.

The sex-determining mechanism of swordtails (Xiphophorus, Poeciliidae, Pisces) is poorly understood, because many laboratory strains of these species are characterized by biased sex ratios in either the male or female direction. Because of these ratios sex determination is said to be controlled by numerous male and female factors scattered over many chromosomes. However, direct experimental evidence for the polygenic theory is absent. Three strains of X. alvarezi were derived from natural populations in Guatemala, two of which (Dolores, Candelaria) always exhibit a ratio of 1:1, whereas the third one (San Ramón) shows a preponderance of males. Males of X. alvarezi, like those of other species of swordtails, develop a caudal appendage or sword at maturity. Chromatophores with carotenoid pigment (orange) are present in the sword of Dolores males and pigment cells containing sepiapterin (green) are present in the swords of males and the other two stocks. F1 and backcross hybrids were produced between the Dolores and Candelaria stocks. The sex ratios of all hybrid pedigrees was always in statistical agreement with unity. The pattern of inheritance of the two pigmentary traits demonstrated sex-linkage with female heterogamety (WY female female-YY male male).

Sex differentiation of somatic tissue in the unsexualised gonad primordia of the embryos of three species of poeciliid fish.

The structure of the gonad primordia is described in the embryos of three species of poeciliid fish. Prior to the sexualisation of the germ cells, a dimorphism exists based upon the gonadal somatic features. This serves as a basis for distinguishing between male and female specimens. Presumptive males are characterised by the presence in at least one of their gonad primordia of a conspicuous stalk whereas in presumptive females, a stalk is completely lacking.

An ovarian regression syndrome in the platyfish, Xiphophorus maculatus.

The highly inbred Coatzacoalcos (Cp) strain of the platyfish, Xiphophorus maculatus, was noted for a high percentage of infertile females (XX). The ovaries of approximately one-quarter of all females regress. The time of gonadal atrophy varied from before sexual maturation up to 11 months of age. The gonadotropic zone of the pituitary was hypertrophied in regressed females. Transplants of immature testes and ovarian tissue into the caudal musculature of regressed females and the subsequent maturation of the grafts demonstrated that the ovarian degeneration was not due to pituitary or hypothalamic malfunction or an autoimmune disease. The cause of the gonadal degeneration was apparently localized to the ovary itself. This phenomenon was never observed in males (XY). Regressed ovaries fell into two categories, designated types I and II, with all being characterized by the presence of ductlike structures which resembled male efferent ducts, lined by Sertoli cells. Type I ovaries bore a marked similarity to certain mammalian dysgenetic gonads, while type II ovaries contained many proliferating germ cells and could be compared to the human neoplasm termed gonadoblastoma. It is suggested that the physiological lesion responsible for the ovarian regression syndrome involves the processes that control the determination and differentiation of the germ cells similar to those found in human 46,XY gonadal dysgenesis.

Linkage of two enzyme loci in the fish genus Poecilia (Teleostei:Poeciliidae).

The linkage of loci coding for glucose-6-phosphate dehydrogenase (G6PD) and phosphogluconate dehydrogenase (PGD) is described in fish of the genus Poecilia (Teleostei:Poeciliidae) and designated Poecilia linkage group I. These two loci were shown to assort independently from six other informative markers (peptidase S, malate dehydrogenase 2 [soluble], mannose phosphate isomerase, parvalbumin 2, phosphoglucomutase, and glyceraldehyde-3-phosphate dehydrogenase 2) within the limits of the data obtained. Data for the linkage analyses were generated by scoring starch-gel electrophoretic phenotypes of the eight loci in reciprocal backcross hybrids obtained from matings between Poecilia perugiae and P. vittata. The linkage chi 2 for G6PD-PGD locus pairs was significant (P less than 0.001) in all reciprocal backcross hybrid broods (22.7% recombinants in the combined data), indicating linkage in both parental species. The linkage of G6PD and PGD in gene maps of the poeciliid genera Xiphophorus and Poeciliopsis documents homology of this linkage within the family. Linkages in salmonid and centrarchid fishes suggest conservation of this linkage group in most or all teleosts. The six additional indpendently assorting loci have been assigned to independent linkage groups in Xiphophorus; thus, no example of poeciliid linkage group divergence has yet been identified.

Differences in transcription and promoters of Xmrk-1 and Xmrk-2 genes suggest a role for Xmrk-2 in pigment pattern development in the platyfish, Xiphophorus maculatus.

Pigment (macromelanophore) patterns in the platyfish Xiphophorus maculatus are due to a complex pigmentary locus; for example, the spotted-dorsal (Sd) fin pattern is due to the Sd locus. In interspecific backcross hybrids with the swordtail X. helleri, the Sd pattern changes into benign or malignant dorsal fin melanoma as a result of hemi- or homozygous loss of a platyfish regulatory (R) gene, the tumor suppressor gene Diff, that appears to play a role in the final differentiation of macromelanophores. Closely linked to the pigmentary locus is an epidermal growth factor receptor-like gene, Xmrk-2, that has arisen by duplication from the linked Xmrk-1. The transcriptional expression of the Xmrk genes was determined in various tissues including Sd pigment patterns and melanomas of various growth potential using reverse transcription-polymerase chain reaction. While Xmrk-1 expression was found in all tissues examined, Xmrk-2 expression correlated with pigment cell growth. Xmrk-2 was highly expressed in the dorsal fin exhibiting the Sd pattern but drastically reduced in a platyfish mutant which has lost the capacity to form these pigment cells in the dorsal fin. Most interestingly, Xmrk-2 expression increased with the proliferative capacity of the melanomas but declined once melanoma growth ceased. We conclude that Xmrk-2 plays a role in the formation of the pigment pattern cell type, perhaps in proliferation of precursor cells, which, in melanoma, are kept in a proliferative state due to loss of Diff.(ABSTRACT TRUNCATED AT 250 WORDS)

Estimation of nuclear DNA content by flow cytometry in fishes of the genus Xiphophorus.

1. By use of flow cytometry we measured nuclear DNA content in cells from 16 stocks representing 9 species of the genus Xiphophorus. 2. Significant differences were detected between certain stocks and species with respect to DNA content. 3. Male-female differences were apparent in 5 of 7 stocks in which males and females were studied. 4. Estimation of nuclear DNA content is of potential significance in connection with the genetics of sex determination and the study of taxonomic relationships.

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.